Hemipteran-and coleopteran active toxin proteins from Bacillus thuringiensis

ABSTRACT

A novel  Bacillus thuringiensis  crystal protein exhibiting insect inhibitory activity is disclosed. Growth of  Lygus  insects is significantly inhibited by providing the novel crystal protein in  Lygus  insect diet. Polynucleotides encoding the crystal protein, transgenic plants and microorganisms that contain the polynucleotides, isolated peptides derived from the crystal protein, and antibodies directed against the crystal protein are also provided. Methods of using the crystal protein and polynucleotides encoding the crystal protein to control Hemipteran insects are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/914,364, filed Apr. 27, 2007.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

APPENDIX

Not Applicable.

INCORPORATION OF SEQUENCE LISTING

A paper copy of the Sequence Listing and a computer readable form of thesequence listing provided herein, containing the file named“38-21(54839) SEQ LIST”, which is 126976 bytes in size (measured inMS-DOS), and are herein incorporated by reference. This Sequence Listingconsists of SEQ ID NOs:1-59.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the field of insect inhibitoryBacillus thuringiensis proteins and, more particularly, to B.thuringiensis crystal proteins that inhibit hemipteran insects. Isolatedpolynucleotides and proteins, transgenic plants and related methods thatprovide for inhibition of hemipteran insects are described. Alsodescribed are methods for combining the B. thuringiensis crystalproteins that inhibit hemipteran insects with distinct insect controlagents to obtain increased levels of hemipteran insect inhibition,hemipteran insect resistance management, or an expanded spectrum ofinsect pest control.

2. Related Art

Bacillus thuringiensis Crystal Proteins

The Gram-positive soil bacterium Bacillus thuringiensis is well knownfor its production of proteinaceous parasporal crystals, orδ-endotoxins, that are toxic to a variety of Lepidopteran, Coleopteran,and Dipteran larvae. B. thuringiensis produces crystal proteins duringsporulation which are specifically toxic to certain species of insects.Many different strains of B. thuringiensis have been shown to produceinsecticidal crystal proteins, and compositions comprising B.thuringiensis strains which produce proteins having insecticidalactivity have been used commercially as environmentally-acceptableinsecticides because of their toxicity to the specific target insect,and non-toxicity to plants and other non-targeted organisms.

Commercial formulations of naturally occurring B. thuringiensis isolateshave long been used for the biological control of agricultural insectpests. In commercial production, the spores and crystals obtained fromthe fermentation process are concentrated and formulated for foliarapplication according to conventional agricultural practices.

Nomenclature of Crystal Proteins

A review by Hofte et al., (Hofte and Whiteley, Microbiol. Rev.,53:242-255, 1989) describes the general state of the art with respect tothe majority of insecticidal B. thuringiensis strains that have beenidentified which are active against insects of the Order Lepidoptera,i.e., caterpillar insects. This treatise also describes B. thuringiensisstrains having insecticidal activity against insects of the OrdersDiptera (i.e., flies and mosquitoes) and Coleoptera (i.e., beetles). Anumber of genes encoding crystal proteins have been cloned from severalstrains of B. thuringiensis. Hofte et al. (1989) discusses the genes andproteins that were identified in B. thuringiensis prior to 1990, andsets forth the nomenclature and classification scheme which hastraditionally been applied to B. thuringiensis genes and proteins. Cry1genes encode Lepidopteran-toxic Cry1 proteins. Cry2 genes encode Cry2proteins that are toxic to both Lepidopterans and Dipterans. Cry3 genesencode Coleopteran-toxic Cry3 proteins, while Cry4 genes encodeDipteran-toxic Cry4 proteins, etc.

Recently a new nomenclature has been proposed which systematicallyclassifies the Cry proteins based upon amino acid sequence homologyrather than upon insect target specificities. This classification schemeand a comprehensive list of insect inhibitory B. thuringiensis genes issummarized in the listing of Insecticidal Toxin Proteins as set forth inthe Neil Crickmore website accessed through Cambridge University on theworld wide web atlifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/index.html.

Mode of Crystal Protein Toxicity

All δ-endotoxin crystals are toxic to insect larvae by ingestion.Solubilization of the crystal in the midgut of the insect releases theprotoxin form of the δ.-endotoxin which, in most instances, issubsequently processed to an active toxin by midgut protease. Theactivated toxins recognize and bind to the brush-border of the insectmidgut epithelium through receptor proteins. Several putative crystalprotein receptors have been isolated from certain insect larvae(Jurat-Fuentes J L, Adang M J. Biochemistry. 45(32):9688, 2006;Griffitts J S et al., Science. 307(5711):922, 2005; Jurat-Fuentes J L,Adang M J. Eur J. Biochem.; 271(15):3127, 2004). The binding of activetoxins is followed by intercalation and aggregation of toxin moleculesto form pores within the midgut epithelium. This process leads toosmotic imbalance, swelling, lysis of the cells lining the midgutepithelium, and eventual larvae mortality. With the advent of moleculargenetic techniques, various δ.-endotoxin genes have been isolated andtheir DNA sequences determined. These genes have been used to constructcertain genetically engineered B. thuringiensis products that have beenapproved for commercial use. Recent developments have seen newδ.-endotoxin delivery systems developed, including plants that containand express genetically engineered δ.-endotoxin genes. Control ofLepidopteran and Coleopteran pests in a variety of transgenic cropplants including corn, cotton, potato, tomato and rice that expressδ.-endotoxin genes is well established. Advantages associated withexpression of theδ.-endotoxin genes in crop plants include increasedyields and decreased use of chemical insecticides. The advantages oftransgenic crops that express insect inhibitory δ.-endotoxin genes haslead to widespread use in crops such as corn and cotton.

Unfortunately, the δ.-endotoxin genes that are currently available donot provide for control of all insect pests that plague crop production.In particular, Hemipteran insects still must be controlled by use ofinsecticides in crops where they cause damage. The Hemipteran or“piercing/sucking” insects are especially damaging to plants in thatthey are also known to transmit damaging plant viruses and cause plantsto be more susceptible to bacterial and fungal infection. There is thusa need for additional materials and methods that would permit inhibitionof Hemipteran insect pests in crops. There is also a need to obtainseveral different types of Hemipteran insect control agents withdistinct modes of action for use in transgenic plants as Hemipteraninsect resistance management tools.

Given the need for Hemipteran insect control agents, a variety ofapproaches have been disclosed. U.S. Pat. No. 5,723,440 describes aCyt1Ba1 protein with purported activity against Hemipteran insects.However, Wellman-Desbiens and Cote (J. Econ. Entomol. 98(5):1469-1479.,2005) were unable to confirm this activity with the Hemipteran insectLygus hesperus. U.S. Pat. No. 5,885,963 discloses the use of B.thuringiensis israelensis Cyt toxins that purportedly inhibit Hemipteranpests. More recently, US20060242732 discloses B. thuringiensis crystalproteins with activity against the Hemipteran insect Lygus lineolaris.These proteins are unrelated to the Cyt proteins described in U.S. Pat.Nos. 5,723,440 and 5,885,963.

SUMMARY OF THE INVENTION

It is in view of the above problems that the present invention wasdeveloped.

The invention first relates to an isolated polynucleotide which encodesa TIC807 insect inhibitory protein or an insect inhibitory proteinfragment derived therefrom, wherein the insect inhibitory protein orprotein fragment comprises a polypeptide sequence that has at leastabout 70% sequence identity to a corresponding polypeptide sequencecontained within SEQ ID NO:5. The polynucleotide sequences of theinvention can also encode polypeptide sequences with at least about 80%,90%, 95%, or 100% sequence identity to the corresponding insectinhibitory polypeptide sequence contained within SEQ ID NO:5. In certainembodiments, the polynucleotide encodes the polypeptide sequence of SEQID NO:5.

Isolated polynucleotides of the invention can encode a TIC807 insectinhibitory protein or insect inhibitory protein fragment derivedtherefrom that inhibits a Hemipteran insect, a Heteropteran insect or aHomopteran insect. The Hemipteran insect can be a Lygus insect and theHomopteran insect can be an aphid, a hopper, or a whitefly. The encodedTIC807 insect inhibitory protein or insect inhibitory protein fragmentderived therefrom inhibits Lygus at a Lygus diet concentration of atleast about 5 ppm, 50 ppm, 250 ppm, or 500 ppm (parts per million) ofthe TIC807 protein or protein fragment in the Lygus diet. The TIC807insect inhibitory protein fragment encoded by the polynucleotidecomprises a peptide sequence of at least 250 amino acid residues. Thisisolated polynucleotide encoding the TIC807 protein can be modified forimproved expression in plants compared to the native coding sequence.One embodiment of a TIC807 encoding polynucleotide that is designed forexpression in plants is provided as SEQ ID NO:6. Other embodiments ofTIC807 encoding polynucleotides for expression in plants are provided asSEQ ID NO: 45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49,SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53. In otherembodiments, the polynucleotide designed for expression in plantsencodes a TIC807 protein with an N-terminal chloroplast or plastidtargeting peptide. One embodiment is provided in SEQ ID NO:7 andcomprises a polynucleotide designed for expression of the TIC807 toxinin plants that is also linked in frame to a nucleotide sequence encodinga plastid targeting peptide. The plastid targeting peptide is operablylinked to TIC807 upon expression and functions to direct the insertionof the TIC807 toxin into the plant plastid.

Other isolated polynucleotides of the invention include polynucleotidesthat hybridize under high stringency conditions with either the nativeBacillus thuringiensis TIC807 gene (SEQ ID NO:4) or with the genedesigned for improved plant expression of TIC807, which has an enrichedG+C content (SEQ ID NO:6) compared to the native coding sequence setforth at SEQ ID NO:4. Polynucleotides that hybridize under stringentconditions can be selected from the group consisting of SEQ ID NO:4, SEQID NO:6 and SEQ ID NO:7.

The invention further provides for transgenic plants or plant partsderived therefrom comprising a TIC807 insect inhibitory protein or aninsect inhibitory protein fragment derived therefrom, wherein the TIC807insect inhibitory protein or protein fragment comprises a polypeptidesequence that has at least about 70% sequence identity to acorresponding polypeptide sequence contained within SEQ ID NO:5. Thetransgenic plant or plant part comprises the TIC807 protein or proteinfragment at a concentration from at least about 5 μg to about 250 μg ofthe TIC807 protein or protein fragment per gram fresh weight planttissue, or any amount in between. The TIC807 insect inhibitory proteinfragment encoded by the polynucleotide comprises a peptide sequence ofat least 250 amino acid residues. The transgenic plant part can be acell, a leaf, a stem, a flower, a sepal, a fruit, a root, or a seed.

The invention also provides for transformed host cells comprising apolynucleotide which encodes a TIC807 insect inhibitory protein or aninsect inhibitory protein fragment derived therefrom, wherein the insectinhibitory protein or protein fragment comprises a polypeptide sequencethat has at least about 70% sequence identity to a correspondingpolypeptide sequence contained within SEQ ID NO:5. The TIC807 insectinhibitory protein fragment encoded by the polynucleotide comprises apeptide sequence of at least 250 amino acid residues. The transformedhost cell can be a bacterial cell or a plant cell. Transformed plantcells of the invention can be selected from the group consisting ofbarley, corn, oat, rice, rye, sorghum, turf grass, sugarcane, wheat,alfalfa, banana, broccoli, bean, cabbage, canola, carrot, cassaya,cauliflower, celery, citrus, cotton, a cucurbit, eucalyptus, flax,garlic, grape, onion, lettuce, pea, peanut, pepper, potato, poplar,pine, sunflower, safflower, soybean, strawberry, sugar beet, sweetpotato, tobacco, tomato, ornamental, shrub, nut, chickpea, pigeonpea,millets, hops, and pasture grass plant cells. In certain embodiments,the transformed plant cell is a cotton plant cell. Plants derived fromthe transformed plant host cells, seeds produced from the plants derivedfrom the transformed host cell, and progeny plants from that seed arealso contemplated by the invention. Transformed bacterial host cells ofthe invention can be selected from the group consisting of anAgrobacterium, a Bacillus, an Escherichia, a Salmonella, a Pseudomonas,and a Rhizobium bacterial cell. In certain embodiments, the transformedbacterial cell is a Bacillus thuringiensis cell. Another embodiment ofthe invention relates to a biologically-pure or isolated culture of anEscherichia coli strain SIC8088 harboring vector pIC17040, deposited onMar. 16, 2007 with the Agricultural Research Culture Collection,Northern Regional Research Laboratory (NRRL), Peoria, Ill., USA andhaving Accession No. NRRLB-50030.

The invention further provides methods for controlling Lygus comprisingthe steps of: (a) providing a Lygus inhibitory amount of a TIC807 insectinhibitory protein or an insect inhibitory protein fragment derivedtherefrom, wherein the insect inhibitory protein or protein fragmentcomprises a polypeptide sequence that has at least about 70% sequenceidentity to a corresponding polypeptide sequence contained within SEQ IDNO:5; and (b) contacting the Lygus with the inhibitory amount of thepolypeptide sequence, thereby controlling a Lygus insect. Thepolypeptide sequence used in this method can also have at least about90% or 100% sequence identity to the corresponding a correspondinginsect inhibitory polypeptide sequence contained within SEQ ID NO:5. TheTIC807 insect inhibitory protein fragment encoded by the polynucleotidecomprises a peptide sequence of at least 250 amino acid residues. In oneembodiment of this method, the Lygus inhibitory amount of thepolypeptide sequence can be provided in a Lygus diet in step (a) and theLygus can be contacted in step (b) by permitting the Lygus to feed onthe diet. In a more particular embodiment of the method, the Lygus dietis a transgenic plant. When the Lygus diet of this method is atransgenic plant, the Lygus inhibitory amount of the polypeptidesequence is from at least about 5 μg to about 250 μg per gram freshweight tissue of the transgenic plant. In other embodiments of thismethod, the Lygus inhibitory amount of the polypeptide sequence isprovided in step (a) by spraying a composition comprising thepolypeptide on a plant. The composition used in this embodiment of themethod comprises bacterial cells or bacterial spores that express thepolypeptide. In particular embodiments of the method, the bacterialcells or bacterial spores are Bacillus cells or Bacillus spores. Thecomposition used in this method can also comprise parasporal crystalscontaining the polypeptide. In any of these methods of controllingLygus, the plant can be infested with Lygus.

The invention also provides for isolated oligonucleotides comprising atleast 12 contiguous nucleotides of a sequence contained within thenative Bacillus thuringiensis TIC807 protein encoded by SEQ ID NO:4 orcontained within the complement of SEQ ID NO:4. Such isolatedoligonucleotides are useful for detecting either SEQ ID NO:4 or relatedpolynucleotides that encode insect inhibitory proteins related toTIC807. Isolated oligonucleotides comprising at least 12 contiguousnucleotides of a sequence contained within SEQ ID NO:6, SEQ ID NO: 45,SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53 or contained within thecomplement of SEQ ID NO:6, SEQ ID NO: 45, SEQ ID NO:46, SEQ ID NO:47,SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, orSEQ ID NO:53 are also provided by the invention. These isolatedoligonucleotides are useful for detecting either SEQ ID NO:6, apolynucleotide designed for use in plants encoding a TIC807 protein orrelated polynucleotides that encode TIC807 proteins. Kits for detectionof a polynucleotide sequence in a sample that comprise anoligonucleotide that specifically hybridizes to a polynucleotidesequence of SEQ ID NO:6 or a complement thereof and a controlpolynucleotide that hybridizes to the oligonucleotide are also providedby this invention.

Other embodiments of the invention include compositions comprising atleast two degenerate oligonucleotide primers of at least 12 nucleotides,wherein nucleotide sequences of the degenerate oligonucleotide primersare derived from the polypeptide sequence of SEQ ID NO:5. Theseoligonucleotide primer compositions are useful for detectingpolynucleotide sequences in either plant or bacterial samples thatencode TIC807 proteins.

The invention further provides methods for detecting or isolating apolynucleotide that encodes a TIC807 protein or a TIC807 related proteinin a sample that comprise the steps of: (a) selecting a pair ofdegenerate oligonucleotide primers capable of producing an amplicon,wherein nucleotide sequences of the degenerate oligonucleotide primersare derived from a TIC807 polypeptide sequence of SEQ ID NO:5; (b)producing an amplicon from the polynucleotide sequence in the sample;and (c) detecting or isolating the amplicon, thereby detecting orisolating a polynucleotide that encodes a TIC807 protein or a TIC807related protein in a sample. In this method the detected or isolatedamplicon can encode a polypeptide that has at least 45%, 70%, or 90%sequence identity to TIC807 (SEQ ID NO:5). Other methods for detectingor isolating a polynucleotide that encodes a TIC807 protein in a sampleprovided herein comprise the steps of: (a) selecting a degenerateoligonucleotide or collection of degenerate oligonucleotides, whereinnucleotide sequences of the degenerate oligonucleotide primers arederived from a TIC807 polypeptide sequence of SEQ ID NO:5; (b)hybridizing the degenerate oligonucleotide or collection of degenerateoligonucleotides to the sample; (c) detecting hybridization in thesample to a polynucleotide, thereby detecting polynucleotide thatencodes a TIC807 protein in a sample, and (d) isolating thepolynucleotide detected by hybridization in step (c). In this method,the detected polynucleotide can encode a polypeptide that has at least45%, 70%, or 90% sequence identity to TIC807 (SEQ ID NO:5).

The invention also provides methods for expressing a TIC807 protein in aplant that comprise the steps of: (a) inserting into a plant cell genomea nucleic acid sequence comprising in the 5′ to 3′ direction arecombinant, double-stranded DNA molecule, wherein the recombinant,double-stranded DNA molecule comprises: i. a promoter that functions inthe plant cell; ii. a polynucleotide sequence encoding a polypeptidecomprising a TIC807 insect inhibitory protein or an insect inhibitoryprotein fragment derived therefrom, wherein the insect inhibitoryprotein or protein fragment comprises a polypeptide sequence that has atleast about 70% sequence identity to a corresponding polypeptidesequence contained within SEQ ID NO:5; and iii. a 3′ non-translatednucleotide sequence that functions in the cells of the plant to causepolyadenylation, wherein said promoter, said polynucleotide sequence,and said 3′ non-translated nucleotide sequence are operably linked (b)obtaining a transformed plant cell containing the nucleic acid sequenceof step (a); and (c) regenerating from the transformed plant cell atransformed plant that expresses the TIC807 protein. In this method, thepolynucleotide sequence of step (a) can encode either a TIC807 proteinthat has at least 90% sequence identity to SEQ ID NO:5 or can encode theTIC807 protein of SEQ ID NO:5. This polynucleotide sequence can be SEQID NO:6 or another sequence designed for expression in plants thatencodes a TIC807 protein. Polynucleotide sequences designed forexpression in plants include, but are not limited to, SEQ ID NO:6, SEQID NO: 45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53. The TIC807 insectinhibitory protein fragment encoded by the polynucleotide comprises apeptide sequence of at least 250 amino acid residues. In otherembodiments of this method, the polynucleotide sequence of step (a) thatencodes a TIC807 protein is operably linked to a polynucleotide sequencethat encodes a plastid targeting polypeptide. The polypeptide of SEQ IDNO:8 comprises a TIC807 protein that is operably linked to the plastidtargeting polypeptide that can be used in certain embodiments of thismethod.

The invention further provides recombinant DNA vectors comprising in the5′ to 3′ direction: i. a promoter that functions in the plant cell; ii.a polynucleotide sequence encoding a polypeptide comprising a TIC807insect inhibitory protein or an insect inhibitory protein fragmentderived therefrom, wherein said insect inhibitory protein or proteinfragment comprises a polypeptide sequence that has at least about 70%sequence identity to a corresponding polypeptide sequence containedwithin SEQ ID NO:5; and iii. a 3′ non-translated nucleotide sequencethat functions in the cells of the plant to cause polyadenylation, wherethe promoter, said polynucleotide sequence, and said 3′ non-translatednucleotide sequence are operably linked. In these vectors, thepolynucleotide sequence can also encode either a TIC807 protein that hasat least 90% sequence identity to SEQ ID NO:5 or can encode the TIC807protein of SEQ ID NO:5. The TIC807 insect inhibitory protein fragmentencoded by the polynucleotide comprises a peptide sequence of at least250 amino acid residues. The polynucleotide sequence encoding the TIC807protein can be a sequence that is designed for expression in plants.Polynucleotide sequences designed for expression in plants include, butare not limited to, SEQ ID NO:6, SEQ ID NO: 45, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:52, or SEQ ID NO:53. In other embodiments, the polynucleotidesequence that encodes a TIC807 protein is operably linked to apolynucleotide sequence that encodes a plastid targeting polypeptide. Avector of the invention can comprise a polynucleotide sequence encodesthe polypeptide of SEQ ID NO:8, such as the polynucleotide sequence isSEQ ID NO:7. Vectors of the invention can further comprise apolynucleotide that encodes a selectable marker gene. A selectablemarker gene that confers resistance to AMPA, atrazine, bromoxynil,dalapon, dicamba, glyphosate, hygromycin, methotrexate, neomycin,phosphinotricin, a sulfonylurea or 2,4-D or combinations thereof can beused in the vectors of the invention.

Also provided by this invention are commodity products produced from aplant or seed wherein the commodity product contains a detectable amountof a TIC807 protein or a polynucleotide that encodes a TIC807 protein.This commodity product can be derived from a cotton plant or cottonplant seed, or similarly from corn, rice, wheat, soy, chickpea,pigeonpea, sugarcane, sugarbeet, and the like. For example, when thecommodity product is derived from a cotton plant or cotton plant seed,the commodity product can be lint, oil, meal, or hulls.

The invention also provides a method for controlling at least one insectpest comprising the steps of: (a) providing at least two differentinsect pest inhibitory agents in a composition, the compositioncomprising (i) an insect inhibitory amount of a TIC807 protein and aninsect inhibitory amount of (ii) at least one ribonucleotide sequencethat functions upon ingestion by the insect pest to inhibit a biologicalfunction within the insect pest and/or (iii) an insect inhibitory amountof at least one insect inhibitory protein other than a TIC807 protein;and (b) contacting the insect pest or pests with an inhibitory amount ofthe composition. In this method, the insect pest controlled can be ahemipteran insect, a heteropteran insect or a homopteran insect. Onehemipteran insect controlled by the method is a Lygus insect. Ahomopteran insect controlled by the method is an aphid, a hopper or awhitefly. In this method, a TIC807 insect inhibitory protein or proteinfragment comprises a polypeptide sequence that has at least about 70%sequence identity to a corresponding polypeptide sequence containedwithin SEQ ID NO:5. A TIC807 protein used in the method can alsocomprise a TIC807 insect inhibitory protein fragment of at least 250amino acid residues in length. When a biological function is inhibitedby a ribonucleotide, the biological function within the insect pest inii) can be an essential biological function. The essential biologicalfunction inhibited by the method can be provided by an essential proteinor ribonucleic acid of the insect pest, the predicted function of whichis selected from the group consisting of muscle formation, juvenilehormone formation, juvenile hormone regulation, ion regulation andtransport, digestive enzyme synthesis, maintenance of cell membranepotential, amino acid biosynthesis, amino acid degradation, spermformation, pheromone synthesis, pheromone sensing, antennae formation,wing formation, leg formation, development and differentiation, eggformation, larval maturation, digestive enzyme formation, haemolymphsynthesis, haemolymph maintenance, neurotransmission, cell division,energy metabolism, respiration, and apoptosis. The essential biologicalfunction can be inhibited in Lygus by a ribonucleotide sequence thatcomprises from about 21 to about 5000 contiguous nucleotides exhibitingfrom about 80 to about 100% sequence identity to a nucleotide codingsequence selected from the group consisting of SEQ ID NO:24 through SEQID NO:39. In other embodiments of this method, the one insect inhibitoryprotein other than a TIC807 protein can be derived from Bacillusthuringiensis. This insect inhibitory protein other than a TIC 807protein can be selected from the group consisting of AXMI-027, AXMI-036,AXMI-038, AXMI-018, AXMI-020, AXMI-021, AXMI-010, AXMI-003, AXMI-008,AXMI-006, AXMI-007, AXMI-009, AXMI-014, ET29, ET37, AXMI-004, AXMI-028,AXMI-029, AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014, TIC809,TIC810, TIC812, TIC127 and TIC128. In other embodiments where two Lygusinhibitory proteins other than a TIC807 protein are expressed in theplant, the two Lygus inhibitory proteins can comprise TIC809 and TIC810.In other embodiments of the method, both a first and a second insectpest can be controlled by the composition. In these embodiments of themethod, the second insect pest can be inhibited by either theribonucleotide sequence of the composition or by the protein other thana TIC807 protein of the composition. This second insect pest can be alepidopteran insect pest. The second insect pest can be inhibited by aprotein selected from the group consisting of a Cry1A protein, a Cry1Bprotein, a Cry1C, a Cry1A/Cry1F chimeric protein, and a Cry2Ab protein.In certain embodiments of the method of controlling at least one insectpest, the composition provides for a synergistic insect inhibitoryeffect. In other embodiments of the method of controlling at least oneinsect pest, the composition provides for an additive insect inhibitoryeffect. In the methods of controlling at least one insect pest, thecomposition can be a transgenic plant.

The invention also provides a method for protecting a plant from Lygusinfestation comprising expressing a Lygus inhibitory amount of at leasttwo different Lygus inhibitory agents in the plant, where the Lygusinhibitory agents comprise (i) a Lygus inhibitory amount of a TIC807protein; (ii) a Lygus inhibitory amount of at least one Lygus inhibitoryprotein other than a TIC807 protein and/or (iii) a Lygus inhibitoryamount of at least one ribonucleotide sequence that functions uponingestion by the Lygus to inhibit a biological function within theLygus. This essential biological function in Lygus can be provided by anessential protein or ribonucleic acid of the Lygus, the predictedfunction of which is selected from the group consisting of muscleformation, juvenile hormone formation, juvenile hormone regulation, ionregulation and transport, digestive enzyme synthesis, maintenance ofcell membrane potential, amino acid biosynthesis, amino aciddegradation, sperm formation, pheromone synthesis, pheromone sensing,antennae formation, wing formation, leg formation, development anddifferentiation, egg formation, larval maturation, digestive enzymeformation, haemolymph synthesis, haemolymph maintenance,neurotransmission, cell division, energy metabolism, respiration, andapoptosis. This essential biological function in Lygus can be inhibitedby a ribonucleotide sequence that comprises from about 21 to about 5000contiguous nucleotides exhibiting from about 80 to about 100% sequenceidentity to a nucleotide coding sequence selected from the groupconsisting SEQ ID NO:24 through SEQ ID NO:39. In certain embodiments ofthis method, the Lygus inhibitory protein other than a TIC807 protein isderived from Bacillus thuringiensis. The Lygus inhibitory protein otherthan TIC807 can be selected from the group consisting of AXMI-027,AXMI-036, AXMI-038, AXMI-018, AXMI-020, AXMI-021, AXMI-010, AXMI-003,AXMI-008, AXMI-006, AXMI-007, AXMI-009, AXMI-014, ET29, ET37, AXMI-004,AXMI-028, AXMI-029, AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009,AXMI-014, TIC809, TIC810, TIC812, TIC127 and TIC128. In otherembodiments where two Lygus inhibitory proteins other than a TIC807protein are expressed in the plant, the two Lygus inhibitory proteinscan comprise TIC809 and TIC810. In this method, a TIC807 insectinhibitory protein or protein fragment comprises a polypeptide sequencethat has at least about 70% sequence identity to a correspondingpolypeptide sequence contained within SEQ ID NO:5. A TIC807 protein usedin the method can also comprise a TIC807 insect inhibitory proteinfragment of at least 250 amino acid residues in length. In certainembodiments of this method, expression of the Lygus inhibitory agentsprovides for a synergistic Lygus inhibitory effect. In other embodimentsof this method, expression of the Lygus inhibitory agents provides foran additive Lygus inhibitory effect. By using this method, the plant canbe protected from Lygus hesperus or Lygus lineolaris.

The invention further provides for isolated proteins, wherein theisolated protein comprises a polypeptide sequence of at least 9 aminoacids in length that is contained within SEQ ID NO:5. The isolatedprotein can have a polypeptide sequence at least 12, 16, 32 or 250 aminoacids in length. The isolated protein of at least 32 amino acids inlength can have a polypeptide sequence at least about 80%, 90%, or 95%sequence identity to a corresponding polypeptide sequence containedwithin SEQ ID NO:5. The isolated protein of the invention can be aninsect inhibitory protein when it is at least 250 amino acids in length.The isolated insect inhibitory protein of at least 250 amino acids caninhibit Lygus. In certain embodiments, the isolated insect inhibitoryprotein of at least 250 amino acids inhibits Lygus at a Lygus dietconcentration of the protein of at least about 5 ppm, 50 ppm, 250 ppm,or 500 ppm. The isolated protein can also be the protein of SEQ ID NO:5.The isolated protein can further comprise a carrier protein. Thiscarrier protein can be an albumin or a KLH protein. Isolated proteins ofthe invention can also further comprise a covalent modification selectedfrom the group consisting of an indicator reagent, an amino acid spacer,an amino acid linker, a signal sequence, a chloroplast transit peptidesequence, a vacuolar targeting sequence, a stop transfer sequence, atransmembrane domain, a protein purification ligand, or a combinationthereof.

The invention also provides for antibodies that specifically bind to aTIC807 protein or peptide epitope derived therefrom, where the TIC807protein or epitope comprising at least 9 contiguous amino acids of SEQID NO:5.

The invention further provides kits for detection of a TIC807 protein ina sample that comprises: a) an antibody that specifically binds to aTIC807 protein or peptide epitope derived therefrom, the protein orepitope comprising at least 9 contiguous amino acids of SEQ ID NO:5; andb) a control TIC807 protein or peptide epitope derived therefrom thatcomprises at least 9 contiguous amino acids of SEQ ID NO:5.

Further features and advantages of the present invention, as well as thestructure and operation of various embodiments of the present invention,are described in detail below with reference to the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate the embodiments of the present inventionand together with the description, serve to explain the principles ofthe invention. In the drawings:

FIG. 1 illustrates a Needleman-Wunsch global alignment between TIC807(SEQ ID NO:5) and Cry15Aa (SEQ ID NO:41).

FIG. 2 illustrates the Agrobacterium-mediated plant transformationvector pMON105863 that contains both a plastid targeted TIC807 plantexpression cassette and a neomycin selection cassette within theAgrobacterium border sequences.

FIG. 3 illustrates the Agrobacterium-mediated plant transformationvector pMON105864 that contains both a TIC807 plant expression cassetteand a neomycin selection cassette within the Agrobacterium bordersequences.

FIG. 4 illustrates the Agrobacterium-mediated plant transformationvector pMON78892 that contains both a plastid targeted TIC807 plantexpression cassette and a neomycin selection cassette within theAgrobacterium border sequences.

FIG. 5 illustrates the Agrobacterium-mediated plant transformationvector pMON78893 that contains a TIC807 plant expression cassette, aneomycin selection cassette, and a TIC128 expression cassette within theAgrobacterium border sequences.

FIG. 6 illustrates the ClustalW comparison of TIC807 (SEQ ID NO:5) andCry51Aa1 (SEQ ID NO:59).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

As used herein, the phrase “additive effect”, in reference to insectinhibition, refers to an inhibitory effect obtained by combining atleast two distinct insect inhibitory agents that is either: a)quantitatively equivalent to the predicted additive effect of thecombination of the two agents and/or is b) qualitatively equivalent tothe combination of effects obtained from each agent administered on itsown. Examples of quantitative effects include, but are not limited to,changes in LC₅₀, EC₅₀, IC₅₀, percent mortality, or percent stuntingvalues indicative of increased insect inhibitory activity against aknown insect target of both insect inhibitory agents. Examples ofadditive qualitative effects include, but are not limited, to anexpanded spectrum of insect inhibition (i.e., hemipteran andlepidopteran insects) that reflects the simple combination of thespectrum exhibited by each insect inhibitory agent (i.e., thecombination of hemipteran insect inhibition provided by one agent andlepidopteran insect inhibition provided by another agent).

The phrase “Consensus sequence” as used herein refers to an amino acid,DNA or RNA sequence created by aligning two or more homologous sequencesand deriving a new sequence that represents the common amino acid, DNAor RNA sequence.

The term “Construct” as used herein refers to any recombinantpolynucleotide molecule such as a plasmid, cosmid, virus, autonomouslyreplicating polynucleotide molecule, phage, or linear or circularsingle-stranded or double-stranded DNA or RNA polynucleotide molecule,derived from any source, capable of genomic integration or autonomousreplication, comprising a polynucleotide molecule where one or morepolynucleotide molecule has been linked in a functionally operativemanner, i.e., operably linked.

The phrase “biological functional equivalents” as used herein refers topeptides, polypeptides and proteins that contain a sequence orstructural feature similar to a TIC807 protein of the present invention,and which exhibit the same or similar insect inhibitory activity of aTIC807 protein of the present invention. Biological functionalequivalents also include peptides, polypeptides and proteins that reactwith (i.e., specifically bind) to monoclonal and/or polyclonalantibodies raised against a TIC807 protein and that exhibit the same orsimilar insect inhibitory activity as a TIC807 protein.

The phrase “DNA construct” as used herein refers to any DNA molecule inwhich two or more ordinarily distinct DNA sequences have been covalentlylinked. Examples of DNA constructs include but are not limited toplasmids, cosmids, viruses, BACs (bacterial artificial chromosome), YACs(yeast artificial chromosome), plant minichromosomes, autonomouslyreplicating sequences, phage, or linear or circular single-stranded ordouble-stranded DNA sequences, derived from any source, that are capableof genomic integration or autonomous replication. DNA constructs can beassembled by a variety of methods including, but not limited to,recombinant DNA techniques, DNA synthesis techniques, PCR (PolymeraseChain Reaction) techniques, or any combination of techniques.

The phrase “a heterologous promoter”, as used herein in the context of aDNA construct, refers to either: i) a promoter that is derived from asource distinct from the operably linked structural gene or ii) apromoter derived from the same source as the operably linked structuralgene, where the promoter's sequence is modified from its original form.The phrase “high stringency hybridization conditions” refers to nucleicacid hybridization conditions comprising a salt concentration of about1×SSC, a detergent concentration of about 0.1% SDS, and a temperature ofabout 50° C., or equivalents thereof. The term “homolog” as used hereinrefers to a gene related to a second gene by identity of either the DNAsequences or the encoded protein sequences. Genes that are homologs canbe genes separated by the event of speciation (see “ortholog”). Genesthat are homologs may also be genes separated by the event of geneticduplication (see “paralog”). Homologs can be from the same or adifferent organism and may perform the same biological function ineither the same or a different organism.

The term “insect” as used herein refers to any embryonic, larval, nymphor adult form of an Arachnid, Coleopteran, Ctenophalides, Dipteran,Hemipteran, Homopteran, Heteropteran, Hymenopteran or Lepidopteraninsect.

The phrase “an insect inhibitory amount”, refers to an amount of aTIC807 polypeptide, a ribonucleotide, or a protein other than a TIC807protein that results in any measurable inhibition of insect growth,insect development, insect reproduction, insect feeding behavior, insectmating behavior and/or any measurable decrease in the adverse effectscaused by insect feeding on a plant. Similarly, a “Lygus inhibitoryamount” refers to an amount of a TIC807 polypeptide, a ribonucleotide,or a protein other than a TIC807 protein that results in any measurableinhibition of Lygus growth, Lygus development, Lygus reproduction, Lygusfeeding behavior, Lygus mating behavior and/or any measurable decreasein the adverse effects caused by Lygus feeding on a plant.

As used herein when referring to an “isolated DNA molecule”, it isintended that the DNA molecule be one that is present, alone or incombination with other compositions, but not within its naturalenvironment. For example, a coding sequence, intron sequence,untranslated leader sequence, promoter sequence, transcriptionaltermination sequence, and the like, that are naturally found within theDNA of a plant genome are not considered to be isolated from the plantgenome so long as they are within the plant genome from which it wasfirst observed. However, each of these components, and subparts of thesecomponents, would be “isolated” within the scope of this disclosure solong as the structures and components are not within the plant genome.Similarly, a nucleotide sequence encoding a Bacillus thuringiensisinsecticidal protein or any insecticidal variant of that protein wouldbe an isolated nucleotide sequence so long as the nucleotide sequencewas not within the DNA of the Bacillus thuringiensis bacterium fromwhich the structure was first observed. An artificial nucleotidesequence encoding the same amino acid sequence or a substantiallyidentical amino acid sequence that the native B. thuringiensisnucleotide sequence encodes would be considered to be isolated for thepurposes of this disclosure. For the purposes of this disclosure, anytransgenic nucleotide sequence, i.e., the nucleotide sequence of the DNAinserted into the genome of the cells of a plant would be considered tobe an isolated nucleotide sequence whether it is present within theplasmid used to transform plant cells from which transgenic event arose,within the genome of the transgenic event, present in detectable amountsin tissues, progeny, biological samples or commodity products derivedfrom the transgenic event. The nucleotide sequence or any fragmentderived therefrom would therefore be considered to be isolated orisolatable if the DNA molecule can be extracted from cells, or tissues,or homogenate from a plant or seed or plant organ; or can be produced asan amplicon from extracted DNA or RNA from cells, or tissues, orhomogenate from a plant or seed or plant organ, any of which is derivedfrom such materials derived from the transgenic event.

The phrase “ribonucleotide sequence that functions upon ingestion by theinsect pest to inhibit a biological function” refers to RNA sequencethat comprises a sequence that is substantially homologous to an RNAmolecule encoded by a nucleotide sequence within the genome of theinsect, that provides for inhibition of the insect.

As used herein, the term “substantially homologous” or “substantialhomology”, with reference to a nucleic acid or polypeptide sequence,refers to a nucleotide or polypeptide sequence that has about 65% toabout 70% sequence identity, or more preferably from about 80% to about85% sequence identity, or most preferable from about 90% to about 95%sequence identity, to about 99% or 100% sequence identity, with anothernucleotide or polypeptide sequence.

As used herein, the phrase “synergistic effect”, in reference to insectinhibition, refers to an inhibitory effect obtained by combining atleast two distinct insect inhibitory agents that is either: a)quantitatively greater than the predicted additive effect of thecombination of the two agents and/or is b) qualitatively distinct fromany effects obtained from either agent administered on its own. Examplesof quantitative effects include, but are not limited to, changes inLC₅₀, EC₅₀, IC₅₀, percent mortality, or percent stunting valuesindicative of increased insect inhibitory activity against a knowninsect target of both insect inhibitory agents. Examples of synergisticqualitative effects include, but are not limited to, an expandedspectrum of insect inhibition (i.e., hemipteran, homopteran, andlepidopteran insects inhibition) that does not reflect the simplecombination of the spectrum exhibited by each insect inhibitory agentalone (i.e., the combination of hemipteran insect inhibition provided byone agent and lepidopteran insect inhibition provided by another agent).

The phrase “TIC807 protein” as used herein refers to an insectinhibitory protein of at least 250 amino acids that display at least 70%sequence identity to a corresponding polypeptide sequence containedwithin SEQ ID NO:5.

The phrase “TIC807 related protein” as used herein refers to an insectinhibitory protein of at least 250 amino acids that display at least 45%sequence identity to a corresponding polypeptide sequence containedwithin SEQ ID NO:5.

The phrase “operably linked” as used herein refers to the joining ofnucleic acid sequences such that one sequence can provide a requiredfunction to a linked sequence. In the context of a promoter, “operablylinked” means that the promoter is connected to a sequence of interestsuch that the transcription of that sequence of interest is controlledand regulated by that promoter. When the sequence of interest encodes aprotein and when expression of that protein is desired, “operablylinked” means that the promoter is linked to the sequence in such a waythat the resulting transcript will be efficiently translated. If thelinkage of the promoter to the coding sequence is a transcriptionalfusion and expression of the encoded protein is desired, the linkage ismade so that the first translational initiation codon in the resultingtranscript is the initiation codon of the coding sequence.Alternatively, if the linkage of the promoter to the coding sequence isa translational fusion and expression of the encoded protein is desired,the linkage is made so that the first translational initiation codoncontained in the 5′ untranslated sequence associated with the promoterand is linked such that the resulting translation product is in framewith the translational open reading frame that encodes the proteindesired. Nucleic acid sequences that can be operably linked include, butare not limited to, sequences that provide gene expression functions(i.e., gene expression elements such as promoters, 5′ untranslatedregions, introns, protein coding regions, 3′ untranslated regions,polyadenylation sites, and/or transcriptional terminators), sequencesthat provide DNA transfer and/or integration functions (i.e., T-DNAborder sequences, site specific recombinase recognition sites, integraserecognition sites), sequences that provide for selective functions(i.e., antibiotic resistance markers, biosynthetic genes), sequencesthat provide scoreable marker functions (i.e., reporter genes),sequences that facilitate in vitro or in vivo manipulations of thesequences (i.e., polylinker sequences, site specific recombinationsequences) and sequences that provide replication functions (i.e.,bacterial origins of replication, autonomous replication sequences,centromeric sequences).

As used herein, the phrases or terms “sequence identity”, “sequencesimilarity” or “homology” is used to describe sequence relationshipsbetween two or more nucleotide sequences. The percentage of “sequenceidentity” between two sequences is determined by comparing two optimallyaligned sequences over a comparison window, wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) as compared to the reference sequence (which does notcomprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison, and multiplying the result by 100to yield the percentage of sequence identity. A sequence that isidentical at every position in comparison to a reference sequence issaid to be identical to the reference sequence and vice-versa. A firstnucleotide sequence when observed in the 5′ to 3′ direction is said tobe a “complement” of, or complementary to, a second or referencenucleotide sequence observed in the 3′ to 5′ direction if the firstnucleotide sequence exhibits complete complementarity with the second orreference sequence. As used herein, nucleic acid sequence molecules aresaid to exhibit “complete complementarity” when every nucleotide of oneof the sequences read 5′ to 3′ is complementary to every nucleotide ofthe other sequence when read 3′ to 5′. A nucleotide sequence that iscomplementary to a reference nucleotide sequence will exhibit a sequenceidentical to the reverse complement sequence of the reference nucleotidesequence.

As used herein, the phrase “corresponding polypeptide sequence containedwithin SEQ ID NO:5” refers to polypeptide sequence within SEQ ID NO:5that will yield the highest percent identity when aligned with the otherpolypeptide sequence.

As used herein, a “comparison window” refers to a conceptual segment ofat least 6 contiguous positions, usually about 50 to about 100, moreusually about 100 to about 150, in which a sequence is compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. The comparison window may compriseadditions or deletions (i.e., gaps) of about 20% or less as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. Those skilled in the artshould refer to the detailed methods used for sequence alignment in theWisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Drive Madison, Wis., USA) or refer to Ausubel et al.(1998) for a detailed discussion of sequence analysis.

The term “regeneration” as used herein refers to any method of obtaininga whole plant from any one of a seed, a plant cell, a group of plantcells, plant callus tissue, or an excised piece of a plant.

The term “transformation” as used herein refers to a process ofintroducing an exogenous DNA sequence (e.g., a vector, a recombinant DNAmolecule) into a cell or protoplast in which that exogenous DNA isincorporated into a chromosome or is capable of autonomous replication.

The phrase “transgenic plant” refers to a plant or progeny thereofderived from a transformed plant cell or protoplast, wherein the plantDNA contains an introduced exogenous DNA molecule not originally presentin a native, non-transgenic plant of the same species.

The phrases “stabilized RNA”, “stabilized dsRNA”, and “stabilized siRNA”refer to combinations of sense-oriented and anti-sense-oriented,transcribed RNA separated by short sequences that permit formation of ahairpin or stem loop structure in the RNA molecule. The phrase “vasculartissue” as used herein refers to any tissues or cells contained withinthe vascular bundle of a plant, including, but not limited to, phloem,protophloem, metaphloem, xylem, protoxylem, or metaxylem cells ortissues.

The term “vector” as used herein refers to any recombinantpolynucleotide construct that may be used for the purpose oftransformation, i.e., the introduction of heterologous DNA into a hostcell.

II. Polynucleotides of the Invention

A variety of polynucleotides that encode TIC807 insect inhibitoryproteins are contemplated by this invention. Such polynucleotides areuseful for production of TIC807 insect inhibitory proteins in host cellswhen operably linked to suitable promoter, transcription terminationand/or polyadenylation sequences. Such polynucleotides are also usefulas probes for isolating homologous or substantially homologouspolynucleotides that encode TIC807 proteins.

One source of polynucleotides that encode TIC807 is the Bacillusthuringiensis strain which contains the TIC807 polynucleotide of SEQ IDNO:4 that encodes the TIC807 polypeptide of SEQ ID NO:5. Thispolynucleotide sequence was originally isolated from a Bacillusthuringiensis host and is thus suitable for expression of the encodedTIC807 polypeptide in other bacterial hosts. For example, SEQ ID NO:4can be used to express TIC807 protein in bacterial hosts that includebut are not limited to Agrobacterium, a Bacillus, an Escherichia, aSalmonella, a Pseudomonas, and a Rhizobium bacterial host cells. The SEQID NO:4 probes are also useful as probes for isolating homologous orsubstantially homologous polynucleotides that encode TIC807 proteins.Such probes can be used to identify homologous or substantiallyhomologous polynucleotides derived from Bacillus strains.

Polynucleotides that encode TIC807 proteins can also be synthesized denovo from a TIC807 polypeptide sequence. The sequence of thepolynucleotide gene can be deduced from a TIC807 polypeptide sequencethrough use of the genetic code. Computer programs such as“BackTranslate” (GCG™ Package, Acclerys, Inc. San Diego, Calif.) can beused to convert a peptide sequence to the corresponding nucleotidesequence that encodes the peptide. Examples of a TIC807 polypeptidesequences that can be used to obtain corresponding nucleotide encodingsequences include, but are not limited to, the TIC807 polypeptidesequence of SEQ ID NO:5. Furthermore, synthetic TIC807 polynucleotidesequences of the invention can be designed so that they will beexpressed in plants. U.S. Pat. No. 5,500,365 describes a method forsynthesizing plant genes to improve the expression level of the proteinencoded by the synthesized gene. This method relates to the modificationof the structural gene sequences of the exogenous transgene, to causethem to be more efficiently transcribed, processed, translated andexpressed by the plant. Features of genes that are expressed well inplants include elimination of sequences that can cause undesired intronsplicing or polyadenylation in the coding region of a gene transcriptwhile retaining substantially the amino acid sequence of the toxicportion of the insecticidal protein. A similar method for obtainingenhanced expression of transgenes in monocotyledonous plants isdisclosed in U.S. Pat. No. 5,689,052. Exemplary polynucleotide sequencesdesigned for expression of a TIC807 protein in plants include, but arenot limited to, SEQ ID NO:6, SEQ ID NO: 45, SEQ ID NO:46, SEQ ID NO:47,SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, orSEQ ID NO:53.

III. Isolated Oligonucleotides, Kits and Methods for Isolation and/orDetection of Polynucleotides that Encode TIC807 Proteins

Isolated oligonucleotides for identifying, detecting, or isolatingpolynucleotides that encode TIC807 proteins are also provided by thepresent invention.

In one embodiment, the isolated oligonucleotides comprise at least 12contiguous nucleotides of a sequence contained within the Bacillusthuringiensis TIC807 encoding gene of SEQ ID NO:4 or contained withinthe complement of SEQ ID NO:4. Such oligonucleotides can be used inhybridization or PCR based methods for identifying or isolatingpolynucleotides that encode TIC807 proteins from strains of Bacillusthuringiensis. Such oligonucleotides can also be used to confirm thepresence or absence of a TIC807-encoding polynucleotide in a host cell.It is further recognized that the oligonucleotides can be used tomutagenize SEQ ID NO:4 when they comprise additional sequences thatcomprise mismatches to SEQ ID NO:4. Such “mutagenesis” oligos are usefulfor identification of TIC807 variants with enhanced insect inhibitoryactivity.

In another embodiment, the isolated oligonucleotides comprise at least12 contiguous nucleotides of a sequence contained within thepolynucleotide of SEQ ID NO:6 or contained within the complement of SEQID NO:6. The polynucleotide of SEQ ID NO:6 is specifically designed forexpression in transgenic plants and encodes the TIC807 protein of SEQ IDNO:5. In still other embodiments, the isolated oligonucleotides compriseat least 12 contiguous nucleotides of a sequence contained within thepolynucleotide of SEQ ID NO: 45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ IDNO:53 or contained within the complement of SEQ ID NO: 45, SEQ ID NO:46,SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,SEQ ID NO:52, or SEQ ID NO:53. Such oligonucleotides can be used inhybridization or PCR based methods for detecting SEQ ID NO:6, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ IDNO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53 polynucleotides insamples derived from transgenic plants. When the sample is a ribonucleicacid sample, the oligonucleotides can be used in hybridization or PCRbased methods to quantitate levels of TIC807 transgene expression. Whenthe sample is a deoxyribonucleic acid sample, the oligonucleotides canbe used in hybridization or PCR based methods to determine the presenceor absence of the TIC807 transgene in the sample. It is also anticipatedthat the SEQ ID NO:6 derived oligonucleotides can be used to determinethe presence or absence of a TIC807 transgene in a deoxyribonucleic acidsample derived from a commodity product. Given the exquisite sensitivityof certain nucleic acid detection methods that employ oligonucleotides,it is anticipated that the SEQ ID NO:6 derived oligonucleotides can alsobe used to detect a TIC807 transgene in commodity products derived frompooled sources where only a fraction of the commodity product is derivedfrom a transgenic plant containing SEQ ID NO:6. It is further recognizedthat the oligonucleotides can be used to mutagenize SEQ ID NO:6 whenthey comprise additional sequences that comprise mismatches to SEQ IDNO:6. Such “mutagenesis” oligonucleotides are useful for identificationof TIC807 variants with enhanced insect inhibitory activity and/orenhanced expression in transgenic plant host cells.

It is of course understood that the oligonucleotides of the inventioncan further comprise additional sequences that are not identical orcomplementary to the polynucleotide sequences that encode TIC807proteins. Additional sequences may include but are not limited tosequences used as adapters that facilitate cloning, mutagenesis, ordetection. The oligonucleotides of the invention can further compriseadditional covalent modifications. Covalent modifications would include,but are not limited to, detectable labels such as isotopes,fluorophores, and haptens. Biotin is one particularly useful hapten.

Kits for detection of a TIC807 polynucleotide sequence in a sample thatcomprise at least one oligonucleotide that specifically hybridizes tothe polynucleotide sequence of SEQ ID NO:6, SEQ ID NO: 45, SEQ ID NO:46,SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,SEQ ID NO:52, SEQ ID NO:53 or a complement thereof are furthercontemplated by this invention. In the context of the kits of thisinvention, the term “specifically hybridize” means that theoligonucleotides will hybridize and detect SEQ ID NO:6, SEQ ID NO: 45,SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53 in a sample from atransgenic plant transformed with one or more copies of SEQ ID NO:6, SEQID NO: 45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53 but will notspecifically hybridize and detect any sequences in a controlnon-transgenic plant that does not contain SEQ ID NO:6, SEQ ID NO: 45,SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53. These kits can alsocomprise a control polynucleotide that hybridizes to saidoligonucleotide, instructions for use, and/or reagents for hybridizingor detecting hybridization of the oligonucleotides to SEQ ID NO:6, SEQID NO: 45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53. In certainapplications, including but not limited to those application that use aPolymerase Chain Reaction, the kits will naturally comprise more thanone oligonucleotide that specifically hybridizes to the polynucleotidesequence of SEQ ID NO:6, SEQ ID NO: 45, SEQ ID NO:46, SEQ ID NO:47, SEQID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQID NO:53.

IV. Degenerate Oligonucleotides, Degenerate Oligonucleotide Compositionsand Methods of Use

Degenerate oligonucleotides, compositions comprising degenerateoligonucleotides, and methods of using such oligonucleotides toidentify, detect or isolate TIC807 protein encoding polynucleotides arealso contemplated by this invention. Although such degenerateoligonucleotides are derived from SEQ ID NO:5, those skilled in the artappreciate that such oligonucleotides can be used to identify a varietyof TIC807 proteins and TIC807-related proteins. Such TIC807 proteins areanticipated to have at least 70%, 80%, 90%, 95%, 98% or 100% amino acididentity to SEQ ID NO:5 and to have insect inhibitory activity. TheTIC807 related proteins have at least 45% sequence identity to SEQ IDNO:5 and have insect inhibitory activity.

The design of degenerate oligonucleotides sequences from peptidesequences is accomplished through use of the genetic code, wherebycodons corresponding to each of the encoded amino acids are synthesized.Degenerate oligonucleotides can comprise either pool of oligonucleotidescomprising all of the potential sequences that encode a given peptidesequence. Alternatively, the degenerate oligonucleotides can alsocomprise a sequence that contains a neutral base (i.e., a base that canbase pair adequately with all nucleotides at a given position). Neutralbases include, but are not limited to, inosine. Considerations involvedin the design and use of degenerate oligonucleotide primers or probesare well known to those skilled in the art (see Molecular Cloning: ALaboratory Manual (Third Edition), Sambrook and Russell, Cold SpringHarbor Press, 2001).

This invention discloses and claims compositions comprising at least twodegenerate oligonucleotide primers of at least 12 nucleotides from thepolypeptide sequence of SEQ ID NO:5. Such compositions can be used ineither hybridization or polymerase chain reaction based methods forisolation or detection of polynucleotides that encode TIC807 proteins orTIC807 related proteins. The degenerate oligonucleotides of thiscomposition can further comprise additional sequences that are notidentical or complementary to the polynucleotide sequences that encodeTIC807 proteins. Additional sequences may include but are not limited tosequences used as adapters that facilitate cloning, mutagenesis, ordetection. The degenerate oligonucleotides of the invention can furthercomprise additional covalent modifications. Covalent modifications wouldinclude, but are not limited to, detectable labels such as an isotopes,fluorophores, and haptens. Biotin is one particularly useful hapten.

Use of the degenerate oligonucleotide primers in PCR based methods ofisolating or detecting polynucleotides that encode a TIC807 protein or aTIC807 related protein in a sample is specifically contemplated. Inbrief, a pair of degenerate oligonucleotide primers capable of producingan amplicon is selected and used in a polymerase chain reaction with asample that contains a polynucleotide that encodes a TIC807 protein or aTIC807 related protein. A suitable source of samples for this methodinclude, but are not limited to, various Bacillus thuringiensis strains.The degenerate oligonucleotides are capable of producing an ampliconwhen the oligonucleotides correspond to predicted sense and antisensestrand sequences and are in a 5′ to 3′ orientation that will prime DNApolymerase-mediated synthesis of a DNA strand that is complementary tothe other opposing oligonucleotide. The degenerate oligonucleotideprimers are derived from a TIC807 polypeptide sequence of SEQ ID NO:5.This amplicon can be detected by use of an intercalating dye to producean amplicon. The amplicon can also be isolated by cloning the isolatedamplicon fragment into a plasmid, cosmid, bacteriophage, or othercloning vector. Once cloned, this amplicon can be further characterizedby sequencing to determine the percent identity of the amplicon-encodedprotein to TIC807 (SEQ ID NO:5). It is anticipated that polynucleotidesencoding TIC807 proteins of at least 70% or at least 90% identity to SEQID NO:5 and TIC807-related proteins of at least 45% identity to SEQ IDNO:5 can be detected or isolated by these methods. Such TIC807 proteinsor a TIC807 related proteins can subsequently be screened for insectinhibitory activity.

The degenerate TIC807 oligonucleotides can also be used as probes inhybridization based methods of detecting or isolating polynucleotidesthat encode TIC807 proteins or a TIC807 related proteins. Methods fordetecting a polynucleotide that encodes a TIC807 protein in a samplefirst comprise selecting a degenerate oligonucleotide or collection ofdegenerate oligonucleotide derived from a TIC807 polypeptide sequence ofSEQ ID NO:5. These degenerate oligonucleotides may further comprisedetectable labels such as isotopes, fluorophores, and haptens. Biotin isone particularly useful hapten. The samples include, but are not limitedto, samples derived from various Bacillus thuringiensis strains. Thesample can be a library of plasmid, cosmid or bacteriophage clonesderived from one or more Bacillus thuringiensis strains. The degenerateoligonucleotide or collection of degenerate oligonucleotides arehybridized to the sample under suitable hybridization stringencyconditions. These conditions are related to the length of the degenerateoligonucleotide(s), the degree of degeneracy, their G+C content, thedesired or projected percent sequence identity of target sequences inthe sample and other factors. Hybridization to a polynucleotide isdetected by methods including, but not limited to, radiometric,fluorometric, luminometric, and/or ELISA-based methods. Followingdetection, the polynucleotide can be isolated by serial dilution andre-hybridization. All of the above listed steps of degenerateoligonucleotide design, oligonucleotide labeling, library preparation,hybridization, detection and isolation are well know to those skilled inthe art (see Molecular Cloning: A Laboratory Manual (Third Edition),Sambrook and Russell, Cold Spring Harbor Press, 2001). It is anticipatedthat polynucleotides encoding TIC807 proteins of at least 70% or atleast 90% identity to SEQ ID NO:5 and TIC807-related proteins of atleast 45% identity to SEQ ID NO:5 can be detected or isolated by thesemethods. Such TIC807 proteins or a TIC807 related proteins cansubsequently be screened for insect inhibitory activity followingexpression in an acrystallifeorus Bacillus thuringiensis strain. TheTIC807 or TIC807 related proteins can inhibit a hemipteran pest such asLygus. Alternatively, the TIC807 or TIC807 related proteins can inhibitother insect pests including Arachnid, Coleopteran, Ctenophalides,Dipteran, Hymenopteran or Lepidopteran pests, or can inhibit bothhemipteran pests and other families of insect pests.

V. DNA Constructs Comprising TIC807 Bacterial Expression Cassettes

To express TIC807 proteins in bacterial hosts, polynucleotides thatencode TIC807 are operably linked to suitable promoters andtranscriptional termination sequences that function in bacterial hoststo yield bacterial expression cassettes. Promoters and terminationsignals that function in bacterial cells can be derived from bacterialgenes, bacteriophage genes or synthetic methods. These expressioncassettes can then be transferred to suitable bacterial vectors thatcomprise replication origins and selectable markers via standardrecombinant DNA techniques.

In the practice of this invention, bacterial promoters, terminationsignals and vectors that function in Bacillus hosts are particularlyuseful for expression of TIC807 polypeptides. In many instances, theTIC807 gene comprising its endogenous promoter and termination sequencescan be used for expression of TIC807 proteins in Bacillus host cellsthat include but are not limited to Bacillus thuringiensis hosts. Forsuch experiments, use of a shuttle vector that functions in both E. coliand Bacillus hosts is particularly useful. Examples of such shuttlevectors include, but are not limited to, vectors such as pEG854described in U.S. Pat. No. 5,650,308. These shuttle vectors includeantibiotic resistance marker genes permitting transformation of Bacillushosts. Preferred Bacillus thuringiensis hosts include, but are notlimited to, acrystalliferous (Cry protein deficient) B. thuringiensishost strains such as EG10368 and EG10650 (described in U.S. Pat. No.5,759,538). When the TIC807 protein is expressed in a acrystalliferous(Cry protein deficient) B. thuringiensis host strains, the TIC807protein is easily isolated as a parasporal crystal following inductionof sporulation in the host cells. This facile Bacillus thuringiensisexpression system can thus be used to test large numbers of TIC807protein variants for insect inhibitory activity.

VI. DNA Constructs Comprising TIC807 Plant Expression Cassettes

The construction of expression cassettes for use in monocotyledonousplants or dicotyledonous plants is well established. Expressioncassettes are DNA constructs where various promoter, coding, andpolyadenylation sequences are operably linked. In general, expressioncassettes typically comprise a promoter that is operably linked to asequence of interest which is operably linked to a polyadenylation orterminator region. In certain instances including, but not limited to,the expression of transgenes in monocot plants, it may also be useful toinclude an intron sequence. When an intron sequence is included, it istypically placed in the 5′ untranslated leader region of the transgene.In certain instances, it may also be useful to incorporate specific 5′untranslated sequences in a transgene to enhance transcript stability orto promote efficient translation of the transcript.

A variety of promoters can be used in the practice of this invention.One broad class of useful promoters is referred to as “constitutive”promoters in that they are active in most plant organs throughout plantdevelopment. For example, the promoter can be a viral promoter such as aCaMV35S or FMV35S promoter. The CaMV35S and FMV35S promoters are activein a variety of transformed plant tissues and most plant organs (e.g.,callus, leaf, seed and root). Enhanced or duplicate versions of theCaMV35S and FMV35S promoters are particularly useful in the practice ofthis invention (U.S. Pat. No. 5,378,619). Other useful nopaline synthase(NOS) and octopine synthase (OCS) promoters (which are carried ontumor-inducing plasmids of A. tumefaciens), the cauliflower mosaic virus(CaMV) 19S promoters, a maize ubiquitin promoter, the rice Act1 promoterand the Figwort Mosaic Virus (FMV) 35S promoter (see e.g., U.S. Pat. No.5,463,175). It is understood that this group of exemplary promoters isnon-limiting and that one skilled in the art could employ otherpromoters that are not explicitly cited here in the practice of thisinvention.

Promoters that are active in certain plant tissues (i.e., tissuespecific promoters) can also be used to drive expression of TIC807proteins or other insect inhibitory agents. Since certain hemipteraninsect pests are “piercing/sucking” insect that typically feed byinserting their proboscis into the vascular tissue of host plants,promoters that direct expression of insect inhibitory agents in thevascular tissue of the transgenic plants are particularly useful in thepractice of this invention. Various Caulimovirus promoters, includingbut not limited to the CaMV35S, CaMV19S, FMV35S promoters and enhancedor duplicated versions thereof, typically deliver high levels ofexpression in vascular tissues and are thus useful for expression ofTIC807 proteins or other insect inhibitory agents. Phloem-limitedviruses such as the rice tungro virus (Bhattacharyya-Pakrasi et al.,Plant J. 4[1] 71-79, 1993) and the commelina yellow mottle virus(Medberry et al., Plant Cell 4:185-192, 1992) also contain usefulpromoters that are active in vascular tissues. For control of hemipteraninsects that feed on phloem, phloem cell-specific or phloem-preferredpromoters can be used to express TIC807 proteins or other insectinhibitory agents in phloem of transgenic plants. Examples of usefulphloem specific promoters include, but are not limited to, PP2-type genepromoters (U.S. Pat. No. 5,495,007), sucrose synthase promoters (Yangand Russell, Proc. Natl. Acad. Sci. USA 87:4144-4148, 1990), glutaminesynthetase promoters (Edwards et al., Proc. Natl. Acad. Sci. USA87:3459-3463, 1990), and phloem-specific plasma membrane H+-ATPasepromoters (DeWitt et al., Plant J. 1[1]: 121-128, 1991), prunasinhydrolase promoters (U.S. Pat. No. 6,797,859), and a rice sucrosetransporter (U.S. Pat. No. 7,186,821). For control of hemipteran peststhat feed on xylem tissue, a variety of promoters that are active inxylem tissue including, but not limited to, protoxylem or metaxylem canbe used. Promoters active in xylem tissue include, but are not limitedto, promoters associated with phenylpropanoid biosynthetic pathways,such as the phenylalanine ammonia-lyase (PAL) promoters, cinnamate4-hydroxylase (C4H) promoters, coumarate 3-hydroxylase promoters,O-methyl transferase (OMT) promoters, 4-coumarate:CoA ligase (4CL)promoters (U.S. Pat. No. 6,831,208), cinnamoyl-CoA reductase (CCR)promoters and cinnamyl alcohol dehydrogenase (CAD) promoters.

Transcriptional enhancer elements can also be used in conjunction withany promoter that is active in a plant cell or with any basal promoterelement that requires an enhancer for activity in a plant cell.Transcriptional enhancer elements can activate transcription in variousplant cells and are usually 100-200 base pairs long. The enhancerelements can be obtained by chemical synthesis or by isolation fromregulatory elements that include such elements, and can compriseadditional flanking nucleotides that contain useful restriction enzymesites to facilitate subsequence manipulation. Enhancer elements can betypically placed within the region 5′ to the mRNA cap site associatedwith a promoter, but can also be located in regions that are 3′ to thecap site (i.e., within a 5′ untranslated region, an intron, or 3′ to apolyadenylation site) to provide for increased levels of expression ofoperably linked genes. Enhancer elements can also be multimerized(provided in any finite number of linked copies) to provide forincreased expression of operably linked genes. Multimerized enhancersinclude, but are not limited to, duplicate, triplicate, or quadruplicatecopies of enhancers in any orientation or combination of orientations.Enhancers are often derived from plant viral promoters, particularlythose of the of the double-stranded DNA Culimoviridae group comprisingthe caulimoviruses and the badnaviruses. The plant viral promoters orderived plant viral enhancers can provide strong constitutive expressionof operably linked genes in transgenic plants. Enhancers derived fromfragments of these promoters have been demonstrated to effectivelyenhance the performance of promoters driving the expression oftransgenes in plants. Examples of plant viruses useful for isolatingenhancers include, but are not limited to, the cauliflower mosaic virus(CaMV) (see, e.g., Odel et al., Nature 313:810, 1985), the figwortmosaic virus (U.S. Pat. No. 5,378,619), the carnation etched ring virus(CERV) (Hull et al., (1986) EMBO Journal 5:3083-3090), the cassaya veinmosaic virus (CsVMV) (Calvert et al. (1995) J. Gen. Virol. 76: 1271-1278and U.S. Pat. No. 6,963,021), the mirabilis mosaic virus (MMV) (Dey etal. (1999) Plant Mol Biol. 40:771-82), the Cestrum yellow leaf curlingvirus (CmYLCV) (Stavolone et al. (2003) Plant Mol Biol. 53:663-73), thecotton leaf curl Multan virus (CLCuMV) (Xie et al. (2003) Plant MolBiol. 53:1-14), the commelina yellow mottle virus (COYMV) (U.S. Pat. No.6,963,021) and the peanut chlorotic streak caulimovirus (PCLSV) (U.S.Pat. No. 5,850,019). Duplications of enhancers are used in enhancedversions of the CaMV 35S and FMV 35S promoters

Various 5′ untranslated leader sequences can also be operably linked toa coding sequence of interest in a plant expression cassette. Thus theplant expression cassette can contain one or more 5′ non-translatedleader sequences which serve to increase expression of operably linkednucleic acid coding sequences encoding either TIC807 or other proteinsof interest. Without seeking to be limited by theory, such 5′untranslated leader sequences can increase the translational efficiencyof the resultant mRNA and/or increase the stability of the resultantmRNA to provide increased levels of the operably linked and encodedprotein of interest in the transgenic plant. Examples of other useful 5′leader sequences include, but are not limited to, the dSSU 5′, PetHSP705′, and GmHSP17.9 5′ untranslated leader sequences. A translationalenhancer sequence derived from the untranslated leader sequence from themRNA of the coat protein gene of alfalfa mosaic virus coat protein genecan be placed between the promoter and the gene of interest to increasetranslational efficiency of the operably linked gene of interest (U.S.Pat. No. 6,037,527).

An intron may also be included in the DNA expression construct,especially in instances when the sequence of interest is to be expressedin monocot plants. For monocot plant use, introns such as the maizehsp70 intron (U.S. Pat. No. 5,424,412), the maize ubiquitin intron, theAdh intron 1 (Callis et al., 1987), the sucrose synthase intron (Vasilet al., 1989) or the rice Act1 intron (McElroy et al., 1990) can beused. Dicot plant introns that are useful include introns such as theCAT-1 intron (Cazzonnelli and Velten, 2003), the pKANNIBAL intron(Wesley et al., 2001; Collier et al., 2005), the PIV2 intron (Mankin etal., 1997) and the “Super Ubiquitin” intron (U.S. Pat. No. 6,596,925;Collier et al., 2005) that have been operably integrated intotransgenes. It is understood that this group of exemplary introns isnon-limiting and that one skilled in the art could employ other intronsthat are not explicitly cited here in the practice of this invention.

In other embodiments of the invention, sequences encoding peptides thatprovide for the localization of a TIC807 protein in subcellularorganelles can be operably linked to the sequences that encode theTIC807 polypeptide. TIC807 polypeptides that are operably linked to asignal peptide are expected to enter the secretion pathway and can beretained by organelles such as the endoplasmic reticulum (ER) ortargeted to the vacuole by operably linking the appropriate retention ortargeting peptides to the C-terminus of the TIC807 polypeptide. Examplesof vacuolar targeting peptides include, but are not limited to, a CTPPvacuolar targeting signal from the barley lectin gene. Examples of ERtargeting peptides include, but are not limited to, a peptide comprisinga KDEL amino acid sequence. Without seeking to be limited by theory,localization of TIC807 polypeptides in either the endoplasmic reticulumor the vacuole can provide for desirable properties such as increasedexpression in transgenic plants and/or increased efficacy in inhibitinginsects in transgenic plants.

Localization of TIC807 proteins to plant plastids including, but notlimited to, chloroplasts is specifically contemplated herein. Plastidlocalization is typically accomplished by the operable linkage of achloroplast transit peptide sequence to the N-terminus of the TIC807protein. Chloroplast transit peptides (or CTPs) that can be used tolocalize TIC807 proteins in transgenic plants can be derived fromnuclear encoded plant proteins that are targeted to plastids. Nuclearencoded plant proteins that are targeted to plastids include, but arenot limited to, proteins involved in lipid, starch, or amino acidbiosynthesis, as well as proteins involved in photosynthesis. Specificchloroplast transit peptides that can be used include, but are notlimited to, CTPs from nuclear encoded Granule Bound Starch Synthasegenes, plastidial Fatty Acid Desaturase genes, EPSPS genes, and RUBISCOsmall subunit genes. An exemplary CTP is the Arabidopsis EPSPS CTP. Anexemplary nucleic acid (SEQ ID NO:7) encoding an Arabidopsis EPSPS CTPthat is operably linked to a TIC807 protein is provided herein. Withoutseeking to be limited by theory, localization of TIC807 polypeptides inplastids can provide for desirable properties such as increasedexpression in transgenic plants and/or increased efficacy in inhibitinginsects in transgenic plants. Increased expression of other Bacillusthuringiensis proteins through use of chloroplast targeting peptidessuch as Cry1Bb (U.S. patent application Ser. No. 10/525,318), Cry2Ab(U.S. Pat. No. 6,489,542), and Cry3Bb (U.S. Pat. No. 6,501,009) has beendocumented. Without being limited by theory, increased expression ofTIC807 protein in a transgenic plant can provide for increased levels ofinsect inhibition, an expanded spectrum of insect pest inhibition,and/or an increased degree of insect pest resistance management.

As noted above, the sequence of interest can also be operably linked toa 3′ non-translated region containing a polyadenylation signal. Thispolyadenylation signal provides for the addition of a polyadenylatesequence to the 3′ end of the RNA. The Agrobacterium tumor-inducing (Ti)plasmid nopaline synthase (NOS) gene 3′ and the pea ssRUBISCO E9 gene 3′un-translated regions contain polyadenylate signals and representnon-limiting examples of such 3′ untranslated regions that can be usedin the practice of this invention. It is understood that this group ofexemplary polyadenylation regions is non-limiting and that one skilledin the art could employ other polyadenylation regions that are notexplicitly cited here in the practice of this invention.

The DNA constructs that comprise the plant expression cassettesdescribed above are typically maintained in various vectors. Vectorscontain sequences that provide for the replication of the vector andcovalently linked sequences in a host cell. For example, bacterialvectors will contain origins of replication that permit replication ofthe vector in one or more bacterial hosts. Agrobacterium-mediated planttransformation vectors typically comprise sequences that permitreplication in both E. coli and Agrobacterium as well as one or more“border” sequences positioned so as to permit integration of theexpression cassette into the plant chromosome. Such Agrobacteriumvectors can be adapted for use in either Agrobacterium tumefaciens orAgrobacterium rhizogenes. Selectable markers encoding genes that conferresistance to antibiotics are also typically included in the vectors toprovide for their maintenance in bacterial hosts.

VII. Insect Inhibitory Transgenic Plants and Methods for ObtainingInsect Inhibitory Transgenic Plants

Methods of obtaining a transgenic plant capable of inhibiting insectsare also provided by this invention. First, expression vectors suitablefor expression of the TIC807 protein in various dicot and monocot plantsare introduced into a plant, a plant cell or a plant tissue usingtransformation techniques as described herein. Next a transgenic plantcontaining or comprising the TIC807 expression vector is obtained byregenerating that transgenic plant from the plant, plant cell or planttissue that received the expression vector. The final step is to obtaina transgenic plant that expresses an insect inhibitory amount of theTIC807 polypeptide. Transgenic plants expressing insect inhibitoryamounts of TIC807 proteins contemplated herein include, but not limitedto, barley, corn, oat, rice, rye, sorghum, turf grass, sugarcane, wheat,alfalfa, banana, broccoli, bean, cabbage, canola, carrot, cassaya,cauliflower, celery, citrus, cotton, a cucurbit, eucalyptus, flax,garlic, grape, onion, lettuce, pea, peanut, pepper, potato, poplar,pine, sunflower, safflower, soybean, strawberry, sugar beet, sweetpotato, tobacco, tomato, ornamental, shrub, nut, chickpea, pigeonpea,millets, hops, and pasture grass plants.

TIC807 expression vectors can be introduced into the chromosomes of ahost plant via methods such as Agrobacterium-mediated transformation,Rhizobium-mediated transformation, Sinorhizobium-mediatedtransformation, particle-mediated transformation, DNA transfection, DNAelectroporation, or “whiskers”-mediated transformation. Suitable methodsfor transformation of plants include any method by which DNA can beintroduced into a cell, such as by electroporation as illustrated inU.S. Pat. No. 5,384,253; microprojectile bombardment as illustrated inU.S. Pat. Nos. 5,015,580; 5,550,318; 5,538,880; 6,160,208; 6,399,861;and 6,403,865; Agrobacterium-mediated transformation as illustrated inU.S. Pat. Nos. 5,635,055; 5,824,877; 5,591,616; 5,981,840; and6,384,301; and protoplast transformation as illustrated in U.S. Pat. No.5,508,184, etc. Aforementioned methods of introducing transgenes arewell known to those skilled in the art and are described in U.S. PatentApplication No. 20050289673 (Agrobacterium-mediated transformation ofcorn), U.S. Pat. No. 7,002,058 (Agrobacterium-mediated transformation ofsoybean), U.S. Pat. No. 6,365,807 (particle mediated transformation ofrice), and U.S. Pat. No. 5,004,863 (Agrobacterium-mediatedtransformation of cotton). Through the application of techniques such asthese, the cells of virtually any plant species may be stablytransformed, and these cells developed into transgenic plants. Othertechniques that may be particularly useful in the context of cottontransformation are disclosed in U.S. Pat. Nos. 5,846,797, 5,159,135, and6,624,344; and techniques for transforming Brassica plants in particularare disclosed, for example, in U.S. Pat. No. 5,750,871; and techniquesfor transforming soybean are disclosed in for example in Zhang et al.,1999, and U.S. Pat. No. 6,384,301; and techniques for transforming cornare disclosed in WO9506722. Methods of using bacteria such as Rhizobiumor Sinorhizobium to transform plants are described in Broothaerts, etal., Nature. 2005, 10; 433:629-33. It is further understood that theTIC807 expression vector can comprise cis-acting site-specificrecombination sites recognized by site-specific recombinases, includingCre, Flp, Gin, Pin, Sre, pinD, Int-B13, and R. Methods of integratingDNA molecules at specific locations in the genomes of transgenic plantsthrough use of site-specific recombinases can then be used (U.S. Pat.No. 7,102,055). Those skilled in the art will further appreciate thatany of these gene transfer techniques can be used to introduce theexpression vector into the chromosome of a plant cell, a plant tissue ora plant.

The use of plant transformation vectors comprising two separate T-DNAmolecules, one T-DNA containing the gene or genes of interest (i.e., oneor more insect inhibitory genes of interest) and another T-DNAcontaining a selectable and/or scoreable marker gene are alsocontemplated. In these two T-DNA vectors, the plant expression cassetteor cassettes comprising the gene or genes of interest are containedwithin one set of T-DNA border sequences and the plant expressioncassette or cassettes comprising the selectable and/or scoreable markergenes are contained within another set of T-DNA border sequences. Inpreferred embodiments, the T-DNA border sequences flanking the plantexpression cassettes comprise both a left and a right T-DNA bordersequence that are operably oriented to provide for transfer andintegration of the plant expression cassettes into the plant genome.When used with a suitable Agrobacterium host in Agrobacterium-mediatedplant transformation, the two T-DNA vector provides for integration ofone T-DNA molecule containing the gene or genes of interest at onechromosomal location and integration of the other T-DNA containing theselectable and/or scoreable marker into another chromosomal location.Transgenic plants containing both the gene(s) of interest and theselectable and/or scoreable marker genes are first obtained by selectionand/or scoring for the marker gene(s) and screened for expression of thegenes of interest. Distinct lines of transgenic plants containing boththe marker gene(s) and gene(s) of interest are subsequently outcrossedto obtain a population of progeny transgenic plants segregating for boththe marker gene(s) and gene(s) of interest. Progeny plants containingonly the gene(s) of interest can be identified by any combination ofDNA, RNA or protein analysis techniques. Methods for using two T-DNAvectors have been described in U.S. Pat. Nos. 6,265,638, 5,731,179, U.S.Patent Application Publication No. 2003110532A1, and U.S. PatentApplication Publication No. 20050183170A1.

Methods of introducing plant minichromosomes comprising plantcentromeres that provide for the maintenance of the recombinantminichromosome in a transgenic plant can also be used in practicing thisinvention (U.S. Pat. No. 6,972,197). In these embodiments of theinvention, the transgenic plants harbor the minichromosomes asextrachromosomal elements that are not integrated into the chromosomesof the host plant.

Transgenic plants are typically obtained by linking the gene of interest(i.e., in this case a TIC807 expression cassette) to a selectable markergene, introducing the linked transgenes into a plant cell, a planttissue or a plant by any one of the methods described above, andregenerating or otherwise recovering the transgenic plant underconditions requiring expression of said selectable marker gene for plantgrowth. The selectable marker gene can be a gene encoding a neomycinphosphotransferase protein, a phosphinothricin acetyltransferaseprotein, a glyphosate resistant 5-enol-pyruvylshikimate-3-phosphatesynthase (EPSPS) protein, a hygromycin phosphotransferase protein, adihydropteroate synthase protein, a sulfonylurea insensitiveacetolactate synthase protein, an atrazine insensitive Q protein, anitrilase protein capable of degrading bromoxynil, a dehalogenaseprotein capable of degrading dalapon, a 2,4-dichlorophenoxyacetatemonoxygenase protein, a methotrexate insensitive dihydrofolate reductaseprotein, and an aminoethylcysteine insensitive octopine synthaseprotein. The corresponding selective agents used in conjunction witheach gene can be: neomycin (for neomycin phosphotransferase proteinselection), phosphinotricin (for phosphinothricin acetyltransferaseprotein selection), glyphosate (for glyphosate resistant5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) protein selection),hygromycin (for hygromycin phosphotransferase protein selection),sulfadiazine (for a dihydropteroate synthase protein selection),chlorsulfuron (for a sulfonylurea insensitive acetolactate synthaseprotein selection), atrazine (for an atrazine insensitive Q proteinselection), bromoxinyl (for a nitrilase protein selection), dalapon (fora dehalogenase protein selection), 2,4-dichlorophenoxyacetic acid (for a2,4-dichlorophenoxyacetate monoxygenase protein selection), methotrexate(for a methotrexate insensitive dihydrofolate reductase proteinselection), or aminoethylcysteine (for an aminoethylcysteine insensitiveoctopine synthase protein selection).

Transgenic plants can also be obtained by linking a gene of interest(i.e., in this case an TIC807 expression cassette) to a scoreable markergene, introducing the linked transgenes into a plant cell by any one ofthe methods described above, and regenerating the transgenic plants fromtransformed plant cells that test positive for expression of thescoreable marker gene. The scoreable marker gene can be a gene encodinga beta-glucuronidase protein, a green fluorescent protein, a yellowfluorescent protein, a red fluorescent protein, a beta-galactosidaseprotein, a luciferase protein derived from a luc gene, a luciferaseprotein derived from a lux gene, a sialidase protein, streptomycinphosphotransferase protein, a nopaline synthase protein, an octopinesynthase protein or a chloramphenicol acetyl transferase protein.

When the expression vector is introduced into a plant cell or planttissue, the transformed cells or tissues are typically regenerated intowhole plants by culturing these cells or tissues under conditions thatpromote the formation of a whole plant (i.e., the process ofregenerating leaves, stems, roots, and, in certain plants, reproductivetissues). The development or regeneration of transgenic plants fromeither single plant protoplasts or various explants is well known in theart (Horsch, R. B. et al. 1985). This regeneration and growth processtypically includes the steps of selection of transformed cells andculturing selected cells under conditions that will yield rootedplantlets. The resulting transgenic rooted shoots are thereafter plantedin an appropriate plant growth medium such as soil. Alternatively,transgenes can also be introduced into isolated plant shoot meristemsand plants regenerated without going through callus stage tissue culture(U.S. Pat. No. 7,002,058). When the transgene is introduced directlyinto a plant, or more specifically into the meristematic tissue of aplant, seed can be harvested from the plant and selected or scored forpresence of the transgene. In the case of transgenic plant species thatreproduce sexually, seeds can be collected from plants that have been“selfed” (self-pollinated) or out-crossed (i.e., used as a pollen donoror recipient) to establish and maintain the transgenic plant line.Transgenic plants that do not sexually reproduce can be vegetativelypropagated to establish and maintain the transgenic plant line. As usedhere, transgenic plant line refers to transgenic plants derived from atransformation event where the transgene has inserted into one or morelocations in the plant genome. In a related aspect, the presentinvention also encompasses a seed produced by the transformed plant, aprogeny from such seed, and a seed produced by the progeny of theoriginal transgenic plant, produced in accordance with the aboveprocess. Such progeny and seeds will have an TIC807 protein-encodingtransgene stably incorporated into their genome, and such progeny plantswill inherit the traits afforded by the introduction of a stabletransgene in Mendelian fashion. All such transgenic plants havingincorporated into their genome transgenic DNA segments encoding one ormore TIC807 proteins or polypeptides are aspects of this invention. Itis further recognized that transgenic plants containing the DNAconstructs described herein, and materials derived therefrom, may beidentified through use of PCR or other methods that can specificallydetect the sequences in the DNA constructs.

Once a transgenic plant is regenerated or recovered, a variety ofmethods can be used to identify or obtain a transgenic plant thatexpresses a insect inhibitory amount of TIC807. One general set ofmethods is to perform assays that measure the amount of TIC807 that isproduced. For example, various antibody-based detection methodsemploying antibodies that recognize TIC807 can be used to quantitate theamount of TIC807 produced. Examples of such antibody based assaysinclude, but are not limited to, ELISAs, RIAs, or other methods whereinan TIC807-recognizing antibody is detectably labelled with an enzyme, anisotope, a fluorophore, a lanthanide, and the like. By using purified orisolated TIC807 protein as a reference standard in such assays (i.e.,providing known amounts of TIC807), the amount of TIC807 present in theplant tissue in a mole per gram of plant material or mass per gram ofplant material can be determined. The TIC807 protein will typically beexpressed in the transgenic plant at the level of “parts per million” or“ppm” where microgram levels of TIC807 protein are present in gramamounts of fresh weight plant tissue. In this case, 1 microgram ofTIC807 protein per 1 gram of fresh weight plant tissue would represent aTIC807 concentration of 1 ppm. An insect inhibitory amount of TIC807protein is at least 5 ppm (i.e., 5 μg TIC807 protein per gram freshweight plant tissue). In preferred embodiments, a insect inhibitoryamount of TIC807 protein is at least 50 ppm (i.e., 50 μg TIC807 proteinper gram fresh weight plant tissue). In more preferred embodiments, theamount of TIC807 is at least 250 ppm (i.e. 50 μg TIC807 protein per gramfresh weight plant tissue).

Alternatively, the amount of TIC807 mRNA produced by the transgenicplant can be determined to identify plants that express insectinhibitory amounts of TIC807 protein. Techniques for relating the amountof protein produced to the amount of RNA produced are well known tothose skilled in the art and include methods such as constructing astandard curve that relates specific RNA levels (i.e., TIC807 mRNA) tolevels of the TIC807 protein (determined by immunologic or othermethods). Methods of quantitating TIC807 mRNA typically involve specifichybridization of a polynucleotide to either the TIC807 mRNA or to a cDNA(complementary DNA) or PCR product derived from the TIC807 RNA. Suchpolynucleotide probes can be derived from either the sense and/orantisense strand nucleotide sequences of the TIC807 protein-encodingtransgene. Hybridization of a polynucleotide probe to the TIC807 mRNA orcDNA can be detected by methods including, but not limited to, use ofprobes labelled with an isotope, a fluorophore, a lanthanide, or ahapten such as biotin or digoxigenin. Hybridization of the labelledprobe may be detected when the TIC807 RNA is in solution or immobilizedon a solid support such as a membrane. When quantitating TIC807 RNA byuse of a quantitative reverse-transcriptase Polymerase Chain Reaction(qRT-PCR), the TIC807-derived PCR product can be detected by use of anyof the aforementioned labelled polynucleotide probes, by use of anintercalating dye such as ethidium bromide or SYBR green, or use of ahybridization probe containing a fluorophore and a quencher such thatemission from the fluorophore is only detected when the fluorophore isreleased by the 5′ nuclease activity of the polymerase used in the PCRreaction (i.e., a TaqMan™ reaction; Applied Biosystems, Foster City,Calif.) or when the fluorophore and quencher are displaced by polymerasemediated synthesis of the complementary strand (i.e., Scorpion™ orMolecular Beaconυ probes). Various methods for conducting qRT-PCRanalysis to quantitate mRNA levels are well characterized (Bustin, S.A.; Journal of Molecular Endocrinology 29, 23, 2002). Fluorescent probesthat are activated by the action of enzymes that recognize mismatchednucleic acid complexes (i.e., Invader™, Third Wave, Technologies,Madison, Wis.) can also be used to quantitate RNA. Those skilled in theart will also understand that RNA quantitation techniques such asQuantitative Nucleic Acid Sequence Based Amplification (Q-NASBA™) can beused to quantitate TIC807 protein-encoding mRNA and identify expressingplants.

Transgenic plants that express insect inhibitory amounts of TIC807 canalso be identified by directly assaying such plants for insectinhibition. Since Lygus is a phytophagous, piercing-sucking insect, inplanta expression and testing of toxin proteins must be presented in amanner that will permit feeding by the insect from the plant and itsassociated tissues. Several factors are critical in selecting a plantspecies for transformation that will allow for testing of the toxinproteins. The plant must be easily transformable and the tissue derivedfrom the plant must be of the type that is preferred by the insect pest.For this purpose, it is preferable to use a plant that has leaves orother organs that have a large enough surface area to attach a barrierthat inhibits the mobility of the insect pest and forces the organism tofeed from the plant organ. In addition, the vascular tissue of the plantorgan must be close enough to the surface of the organ to allow for theinsect pest to probe, penetrate and subsequently feed. It is alsopreferable that the plant used in transformation be of the type that caneasily be induced to develop from undifferentiated callus.

Insect pests such as Lygus, when feeding on a cotton plant, typicallyfeed primarily at the flower buds or bolls. Cotton transformation iswell known in the art; however the time it takes to go fromtransformation of plant cells to a fully developed cotton plant is toolong to be practical for screening purposes. Therefore, undifferentiatedcotton callus tissue would be the preferred initial transgenic planttesting material when studying Lygus feeding on cotton cells transformedwith TIC807 proteins. Cotton cells are transformed with constructscontaining the TIC807 protein encoding gene. Callus tissue is allowed todevelop in tissue culture after transformation in a Petri dish. TheLygus nymphs are then placed into the Petri dish containing the callus.The secured lid of the Petri dish prevents the escape of the Lygusnymphs. Any material that will prevent Lygus escape but allow gasexchange in the Petri dish, for example, Parafilm® can be used to securethe Petri dish lid. A percentage of Lygus nymphs will find the callustissue and feed. Scores for mortality and stunting are then calculatedtaking into account the background death that will occur from thoseinsects which fail to feed on the callus tissue. Lygus nymphs would alsobe presented with control callus tissue that is not transformed with aTIC807 encoding gene as a control for normal nymph growth on callustissue.

An alternate tissue for TIC807 protein mediated inhibition is leaftissue. Any plant that possesses leaf tissue with a surface areasufficient to place a barrier preventing Lygus escape could be used. Forexample, alfalfa, corn, soybean or lettuce cells can be transformed withconstructs containing the toxin protein encoding gene or genes ofinterest that have been optimized for monocot or dicot expression. Thetransformed cells are allowed to develop into callus tissue and thensubsequently regenerated into plants. Insect pests such as Lygus nymphsare then allowed to feed when the plant has reached a sufficient levelof maturity, such as when the leaves have grown to a size permitting theuse of a physical barrier to prevent Lygus escape. The barrier toprevent escape of the Lygus nymphs can be any commercially available orhome made device that permits contact of the Lygus nymphs with the leaftissue and allows the insect to probe and feed from the vascular tissueof the leaf. Clip cages similar to those described by Mowry (1993) (J.Agric. Entomol. 10:181-184) would be sufficient to contain the Lygusnymphs for feeding. Mortality and stunting scores are then determinedwith respect to the background death that will occur from those insectswhich fail to feed on the leaf tissue. Lygus nymphs would also bepresented with control leaf that is not transformed with a TIC807encoding gene as a control for normal nymph growth on callus tissue.

The in planta insect inhibition assays can be used to identifytransgenic plants that inhibit any of the large variety of insect peststhat pierce and/or suck the fluids from the cells and tissues of plantsthat must be restricted to the assay tissue. In particular, such insectinhibition assays can be used to test plants expressing TIC807 and/orother insect inhibitory agents. Other insect inhibitory agents include,but are not limited to, i) ribonucleotide sequences that functions uponingestion by said insect pest to inhibit a biological function withinsaid insect and ii) non-TIC807 proteins that are insect inhibitory.These insect pests include those insect pests that pierce and then suckthe phloem sap or cell contents as well as those that macerate the cellsin the vicinity of the feeding zone and then take up the fluid that isreleased from the macerated cells through there proboscis. Insectstargeted by the TIC807 proteins and other insect inhibitory agentsdescribed herein include various hemipteran, homopteran and heteropteraninsects. Inhibition of insects such as Lygus, whiteflies, hoppers andaphids is specifically contemplated by use of TIC807 and other insectinhibitory agents as described herein.

VIII. Transgenic Plant Insect Control Methods

Transgenic plants of the present invention comprising polynucleotidesencoding TIC807 or insecticidal fragments thereof can be used in methodsof controlling insect infestations. Transgenic barley, corn, oat, rice,rye, sorghum, turf grass, sugarcane, wheat, alfalfa, banana, broccoli,bean, cabbage, canola, carrot, cassaya, cauliflower, celery, citrus,cotton, a cucurbit, eucalyptus, flax, garlic, grape, onion, lettuce,pea, peanut, pepper, potato, poplar, pine, sunflower, safflower,soybean, strawberry, sugar beet, sweet potato, tobacco, tomato,ornamental, shrub, nut, chickpea, pigeonpea, millets, hops, and pasturegrass plants can be used in these methods. Transgenic plants such asalfalfa, canola, cotton, lettuce and strawberry plants that are attackedby hemipteran insect pests inhibited by TIC807 proteins are specificallycontemplated by this invention. Even more specifically contemplated bythe present invention are transgenic cotton plants comprisingpolynucleotides encoding TIC807 or insecticidal fragments thereof thatare protected from Lygus species insect infestation. Transgenic plantsof the present invention are particularly effective for controllingspecies of insects that pierce and/or suck the fluids from the cells andtissues of plants, including but not limited to, plant bugs in theMiridae family such as western tarnished plant bugs (Lygus hesperusspecies), tarnished plant bugs (Lygus lineolaris species), and palelegume bugs (Lygus elisus) and stinkbugs (Pentatomidae family species).

Specific types of transgenic plants expressing TIC807 proteins thatinhibit specific insect pests are contemplated by this invention.Transgenic cotton plants expressing TIC807 proteins that inhibitHemipteran insects including Lygus, hoppers and aphids are specificallycontemplated. Transgenic cotton plants that express the TIC807 proteinof SEQ ID NO:5 are anticipated to inhibit Lygus hesperus or Lyguslineolaris. Transgenic alfalfa, canola, lettuce and strawberry plantsthat express the TIC807 protein of SEQ ID NO:5 and that inhibit Lygusare also specifically contemplated.

The transgenic plants expressing insect inhibitory amounts of the TIC807proteins are first identified by any one of the methods describedherein. Initial insect inhibition can be conducted in controlledenvironmental conditions (i.e., in enclosed growth chambers or greenhouses). Transgenic plants can also be subjected to insect infestationin field tests and compared against non-transgenic control plants.Typically, the non-transgenic control plants will include both plantstreated with insecticides and untreated plants. Transgenic plant lines(i.e., transgenic plants derived from distinct transformation eventscomprising transgene insertions into different genomic locations) thatdisplay the best insect inhibitory activity are selected for potentialdevelopment for use in a variety of different genetic backgrounds (i.e.,genetically distinct cultivars, varieties, and/or hybrid germplasms).Methods of introgressing transgenes into distinct germplasms andproducing seed lots that primarily comprise transgenic seed are known tothose skilled in the art. For example, the transgene can be fixed in ahomozygous state in a desired genetic background. Once the transgenefixed in that background, the homozygous transgenic plant can be used toproduce transgenic seed of non-hybrid crops. Alternatively, thehomozygous transgenic plant can be used as a pollen donor or recipientto produce transgenic seed of hybrid crops.

Specific types of transgenic plants expressing TIC807 proteins thatinhibit specific insect pests are contemplated by this invention.Transgenic cotton plants expressing TIC807 proteins that inhibithemipteran insects including Lygus, hoppers and aphids are specificallycontemplated. Transgenic cotton plants that express the TIC807 proteinof SEQ ID NO:5 are anticipated to inhibit Lygus hesperus or Lyguslineolaris. Transgenic alfalfa, canola, and strawberry plants thatexpress the TIC807 protein of SEQ ID NO:5 and that inhibit Lygus arealso specifically contemplated.

IX. Non-Transgenic Control Methods and Compositions

The TIC807 protein compositions disclosed herein will find particularutility as insect inhibitory agents for topical and/or systemicapplication to field crops, grasses, fruits and vegetables, andornamental plants. More specifically, TIC807 can be used in compositionscomprising an insect inhibitory amount of a TIC807 protein composition.In this regard, TIC807 protein compositions made up of TIC807 crystalprotein preparations for Bacillus thuringiensis spores are particularlyuseful. The TIC807 protein composition can comprise the amino acidsequence of SEQ ID NO:5 or to an insect inhibitory protein of at least250 amino acids that displays at least 70% sequence identity to acorresponding polypeptide sequence contained within SEQ ID NO:5.

The insect inhibitory composition can also comprise a Bacillusthuringiensis cell, or a culture of these cells, or a mixture of one ormore B. thuringiensis cells which express one or more of theTIC807crystal proteins of the invention. In certain aspects it may bedesirable to prepare compositions which contain a plurality of crystalproteins, either native or modified, for treatment of one or more typesof susceptible insects.

The inventors contemplate that any formulation methods known to those ofskill in the art may be employed using the proteins disclosed herein toprepare such insect inhibitory compositions. It may be desirable toformulate whole cell preparations, cell extracts, cell suspensions, cellhomogenates, cell lysates, cell supernatants, cell filtrates, or cellpellets of a cell culture (preferably a bacterial cell culture such as aBacillus thuringiensis culture) that expresses one or more TIC807 DNAsegments to produce the encoded TIC807 protein(s) or peptide(s). Themethods for preparing such formulations are known to those of skill inthe art, and may include, e.g., desiccation, lyophilization,homogenization, extraction, filtration, centrifugation, sedimentation,or concentration of one or more cultures of bacterial cells, such asBacillus SIC8091, SIC8092, SIC8093, and SIC8094 cells, which express theTIC807 peptide(s) of interest.

In one embodiment, the insect inhibitory composition comprises an oilflowable suspension comprising lysed or unlysed bacterial cells, spores,or crystals which contain one or more of the novel crystal proteinsdisclosed herein. Preferably the cells are B. thuringiensis cells,however, any such bacterial host cell expressing the novel nucleic acidsegments disclosed herein and producing a crystal protein iscontemplated to be useful, such as Bacillus spp., including B.megaterium, B. subtilis; B. cereus, Escherichia spp., including E. coli,and/or Pseudomonas spp., including P. cepacia, P. aeruginosa, and P.fluorescens. Alternatively, the oil flowable suspension may consist of acombination of one or more of the following compositions: lysed orunlysed bacterial cells, spores, crystals, and/or purified crystalproteins.

In a second embodiment, the insect inhibitory composition comprises awater dispersible granule or powder. This granule or powder may compriselysed or unlysed bacterial cells, spores, or crystals which contain oneor more of the novel crystal proteins disclosed herein. Preferredsources for these compositions include bacterial cells such as B.thuringiensis cells, however, bacteria of the genera Bacillus,Escherichia, and Pseudomonas which have been transformed with a DNAsegment disclosed herein and expressing the crystal protein are alsocontemplated to be useful. Alternatively, the granule or powder mayconsist of a combination of one or more of the following compositions:lysed or unlysed bacterial cells, spores, crystals, and/or purifiedcrystal proteins.

In a third important embodiment, the insect inhibitory compositioncomprises a wettable powder, spray, emulsion, colloid, aqueous ororganic solution, dust, pellet, or collodial concentrate. Such acomposition may contain either unlysed or lysed bacterial cells, spores,crystals, or cell extracts as described above, which contain one or moreof the novel crystal proteins disclosed herein. Preferred bacterialcells are B. thuringiensis cells, however, bacteria such as B.megaterium, B. subtilis, B. cereus, E. coli, or Pseudomonas spp. cellstransformed with a DNA segment disclosed herein and expressing thecrystal protein are also contemplated to be useful. Such dry forms ofthe insecticidal compositions may be formulated to dissolve immediatelyupon wetting, or alternatively, dissolve in a controlled-release,sustained-release, or other time-dependent manner. Alternatively, such acomposition may consist of a combination of one or more of the followingcompositions: lysed or unlysed bacterial cells, spores, crystals, and/orpurified crystal proteins.

In a fourth embodiment, the insect inhibitory composition comprises anaqueous solution or suspension or cell culture of lysed or unlysedbacterial cells, spores, crystals, or a mixture of lysed or unlysedbacterial cells, spores, and/or crystals, such as those described abovewhich contain one or more of the novel crystal proteins disclosedherein. Such aqueous solutions or suspensions may be provided as aconcentrated stock solution which is diluted prior to application, oralternatively, as a diluted solution ready-to-apply.

For these methods involving application of bacterial cells, the cellularhost containing the TIC807 protein gene(s) may be grown in anyconvenient nutrient medium, where the DNA construct provides a selectiveadvantage, providing for a selective medium so that substantially all orall of the cells retain the B. thuringiensis gene. These cells may thenbe harvested in accordance with conventional ways. Alternatively, thecells can be treated prior to harvesting.

When the insecticidal compositions comprise B. thuringiensis cells,spores, and/or crystals containing the modified crystal protein(s) ofinterest, such compositions may be formulated in a variety of ways. Theymay be employed as wettable powders, granules or dusts, by mixing withvarious inert materials, such as inorganic minerals (phyllosilicates,carbonates, sulfates, phosphates, and the like) or botanical materials(powdered corncobs, rice hulls, walnut shells, and the like). Theformulations may include spreader-sticker adjuvants, stabilizing agents,other pesticidal additives, or surfactants. Liquid formulations may beaqueous-based or non-aqueous and employed as foams, suspensions,emulsifiable concentrates, or the like. The ingredients may includerheological agents, surfactants, emulsifiers, dispersants, or polymers.

Alternatively, the TIC807 proteins can be prepared by native orrecombinant bacterial expression systems in vitro and isolated forsubsequent field application. Such protein may be either in crude celllysates, suspensions, colloids, etc., or alternatively may be purified,refined, buffered, and/or further processed, before formulating in anactive biocidal formulation. Likewise, under certain circumstances, itmay be desirable to isolate crystals and/or spores from bacterialcultures expressing the crystal protein and apply solutions,suspensions, or collodial preparations of such crystals and/or spores asthe active bioinsecticidal composition.

Regardless of the method of application, the amount of the activecomponent(s) are applied at an insect inhibitory amount, which will varydepending on such factors as, for example, the specific hemipteran,homopteran, or heteropteran insects to be controlled, the specific plantor crop to be treated, the environmental conditions, and the method,rate, and quantity of application of the insect inhibitory composition.

The insect inhibitory compositions described may be made by formulatingeither the bacterial cell, crystal and/or spore suspension, or isolatedprotein component with the desired agriculturally-acceptable carrier.The compositions may be formulated prior to administration in anappropriate means such as lyophilized, freeze-dried, dessicated, or inan aqueous carrier, medium or suitable diluent, such as saline or otherbuffer. The formulated compositions may be in the form of a dust orgranular material, or a suspension in oil (vegetable or mineral), orwater or oil/water emulsions, or as a wettable powder, or in combinationwith any other carrier material suitable for agricultural application.Suitable agricultural carriers can be solid or liquid and are well knownin the art. The term “agriculturally-acceptable carrier” covers alladjuvants, e.g., inert components, dispersants, surfactants, tackifiers,binders, etc. that are ordinarily used in insecticide formulationtechnology; these are well known to those skilled in insecticideformulation. The formulations may be mixed with one or more solid orliquid adjuvants and prepared by various means, e.g., by homogeneouslymixing, blending and/or grinding the insecticidal composition withsuitable adjuvants using conventional formulation techniques.

The insect inhibitory compositions of this invention are applied to theenvironment of the target hemipteran, homopteran, or heteropteraninsect, typically onto the foliage of the plant or crop to be protected,by conventional methods, preferably by spraying. The strength andduration of insecticidal application will be set with regard toconditions specific to the particular pest(s), crop(s) to be treated andparticular environmental conditions. The proportional ratio of activeingredient to carrier will naturally depend on the chemical nature,solubility, and stability of the insecticidal composition, as well asthe particular formulation contemplated.

Other application techniques, e.g., dusting, sprinkling, soaking, soilinjection, seed coating, seedling coating, spraying, aerating, misting,atomizing, and the like, are also feasible and may be required undercertain circumstances such as e.g., insects that cause root or stalkinfestation, or for application to delicate vegetation or ornamentalplants. These application procedures are also well-known to those ofskill in the art.

The insect inhibitory composition of the invention may be employed inthe method of the invention singly or in combination with othercompounds, including and not limited to other pesticides. The method ofthe invention may also be used in conjunction with other treatments suchas surfactants, detergents, polymers or time-release formulations. Theinsecticidal compositions of the present invention may be formulated foreither systemic or topical use.

The concentration of insect inhibitory agent in the insect inhibitorycomposition which is used for environmental, systemic, or foliarapplication will vary widely depending upon the nature of the particularformulation, means of application, environmental conditions, and degreeof insect inhibitory activity. Typically, the insect inhibitory agent inthe composition will be present in the applied formulation at aconcentration of at least about 1% by weight and may be up to andincluding about 99% by weight. Dry formulations of the compositions maybe from about 1% to about 99% or more by weight of the composition,while liquid formulations may generally comprise from about 1% to about99% or more of the active ingredient by weight. Formulations whichcomprise intact bacterial cells will generally contain from about 10⁴ toabout 10¹² cells/mg of the composition.

The insect inhibitory formulation may be administered to a particularplant or target area in one or more applications as needed, with atypical field application rate per hectare ranging on the order of fromabout 1 g to about 1 kg, 2 kg, 5, kg, or more of active ingredient.

XI. Commodity Products

It is also contemplated that various commodity products may be obtainedwith the compositions and methods of this invention. Moreover, it isspecifically contemplated that one or more advantages can be associatedwith the commodity products derived from this invention. It isanticipated that the use of the TIC807 insect inhibitory protein andassociated methods can provide for commodity products with loweredpesticide residue levels. In certain instances, growers will be promptedto use fewer pesticides such as organophosphates, carbamates,neonicotinoid, and pyrethroid insecticides. Exposure of individuals whogrow, harvest, process or otherwise come into contact with the commodityproducts of this invention to these pesticides is thus anticipated to bereduced. Reduced use of pesticides is also anticipated to provide forreduced costs of commodity product production, reduced levels ofenvironmental contamination and reduced undesirable side effects onbeneficial (non-target) insects and fauna. It is further contemplatedthat the use of this invention will provide for commodity products withlower costs of production due to factors including, but not limited to,increased yield and/or decreased insecticide usage.

XII. Methods of Using TIC807 Insect Inhibitory Proteins in Combinationwith Other Insect Inhibitory Agents

Several methods by which increased resistance to a specific insect pestor broader resistance to several classes of insect pests arecontemplated by this invention. Both methods entail contacting theinsect pest(s) with a TIC807 protein in combination with a distinctinsect inhibitory agent. This distinct insect inhibitory agent caninhibit the same hemipteran insect pests inhibited by the TIC807 toprovide for a decreased incidence of hemipteran insect resistance to theTIC807 protein or other hemipteran insect inhibitory agent.Alternatively, the distinct insect inhibitory agent can inhibit aninsect that is not inhibited by TIC807 to expand the spectrum of insectinhibition obtained.

The potential for insects to develop resistance to certain insecticidesis well documented. Most insect resistance management strategies usinggenetically modified crops expressing insect inhibitory agents rely onthe use of refuge areas that are comprised of crop plants that lack theinsect inhibitory gene. In theory, the refuge provides a region in whichnon-resistant insect populations harboring non-resistant genetic allelesare maintained, lowering the potential for resistance to develop withinthe insect population. However, the refuge strategy suffers from severalshort-comings. First, the growers must accept reduced yields on theacreage planted with the insect inhibitory gene. Second, it is not clearthat refuges will effectively control dominant resistance alleles thatcan arise in the insect population.

An alternative insect resistance management strategy can employtransgenic crops that express two distinct insect inhibitory agents thatoperate through different modes of action. In this case, any insectswith resistance to either one of the insect inhibitory agents will becontrolled by the other insect inhibitory agent, thus reducing thechances of resistance developing in the insect population.

In addition, a single crop may be subject to destruction by severaldifferent classes of insect pests operating at the same time in thefield. For example, a cotton plant can be attacked by both Hemipteranpests, such as Lygus, and Lepidopteran pests such as Spodoptera exigua(beet armyworm), Heliothis zea (cotton bollworm) and/or Helicoverpaarmigera (armyworm) in the course of a growing season. Expression ofdistinct inhibitory agents which are active to each of these pests wouldprovide greater protection to the cotton plant and would increase theyield per acre due to a reduction of loss caused by the insect pests.

A first group of insect inhibitory agents that can be used incombination with a TIC807 protein for insect resistance management orexpanded insect inhibitory spectrum comprise ribonucleotide sequencesthat function upon ingestion by said insect pest to inhibit a biologicalfunction within said insect pest. Specific nucleotide sequences selectedfrom the sequences native to the cells of a particular pest that areinvolved in an essential biological pathway can be expressed in a cellin such a way as to result in the formation of a double stranded RNA, oreven a stabilized double stranded RNA. By inhibiting the essential geneproduct of the target insect pest with the ribonucleotide, the organismfails to develop and eventually dies. The use of such ribonucleotidesequences to control insect pests such as Lygus is described in UnitedStates Patent Application Publication No. 20060021087. Essential insectgenes that provide essential biological function that include, but arenot limited to, muscle formation, juvenile hormone formation, juvenilehormone regulation, ion regulation and transport, digestive enzymesynthesis, maintenance of cell membrane potential, amino acidbiosynthesis, amino acid degradation, sperm formation, pheromonesynthesis, pheromone sensing, antennae formation, wing formation, legformation, development and differentiation, egg formation, larvalmaturation, digestive enzyme formation, haemolymph synthesis, haemolymphmaintenance, neurotransmission, cell division, energy metabolism,respiration, and apoptosis are targeted for inhibition. Insect genesthat can be inhibited include, but are not limited to, genes encoding aV-ATPase protein, a ubiquitin protein, a polyglacturonase protein, apectinase protein, a GABA neurotransmitter transporter protein, a EFIalpha protein, a cytochrome P-450 mono-oxygenase protein, a cuticleprotein precursor protein, a CHD3 protein, and a 20S proteasome protein.The ribonucleotide based insect control agent may also comprisesequences directed against multiple insect target genes. For control ofLygus, inhibitory ribonucleotides directed against SEQ ID NO:24 throughSEQ ID NO:39 or combinations of inhibitory ribonucleotides directedagainst SEQ ID NO:24 through SEQ ID NO:39 are specifically contemplated.The use of SEQ ID NO:24 through SEQ ID NO:39 in the control of insectsis disclosed in United States Patent Application Publication No.20060021087. When multiple insect genes are targeted for suppression, apolycistronic DNA element can be fabricated as illustrated and disclosedin Fillatti, U.S. Application Publication No. 2004-0029283 A1.

A variety of methods can be used to produce inhibitory ribonucleotidesdirected against a target pest in a transgenic plant. In general, theinhibitory dsRNA and the portion of the insect target gene share atleast from about 80% sequence identity, or from about 90% sequenceidentity, or from about 95% sequence identity, or from about 99%sequence identity, or even about 100% sequence identity. Alternatively,the duplex region of the RNA may be defined functionally as a nucleotidesequence that is capable of hybridizing with a portion of the targetgene transcript. A less than full length sequence exhibiting a greaterhomology compensates for a longer less homologous sequence. The lengthof the identical nucleotide sequences may be at least 25, 50, 100, 200,300, 400, 500 or 1000 bases. Normally, a sequence of greater than 20-100nucleotides should be used, though a sequence of greater than about200-300 nucleotides would be preferred, and a sequence of greater than500-1000 nucleotides would be especially preferred depending on the sizeof the target gene.

In another embodiment, the insect inhibitory ribonucleotide can beproduced by an inverted repeat separated by a “spacer sequence”. Thespacer sequence may be a region comprising any sequence of nucleotidesthat facilitates secondary structure formation between each repeat,where this is required. In one embodiment of the present invention, thespacer sequence is part of the sense or antisense coding sequence formRNA. The spacer sequence may alternatively comprise any combination ofnucleotides or homologues thereof that are capable of being linkedcovalently to a nucleic acid molecule. The spacer sequence may comprisea sequence of nucleotides of at least about 10-100 nucleotides inlength, or alternatively at least about 100-200 nucleotides in length,at least about 200-400 nucleotides in length, or at least about 400-500nucleotides in length.

A transgene sequence for producing a dsRNA may comprise a promoter thatis operatively linked to an intron encoding sequence and a hairpin RNAderived from a sequence in the target gene (Miki and Shimamoto, PlantCell Physiol. April 2004; 45(4):490-495). Alternatively, a transgenesequence for producing an siRNA may comprise an RNA pol III promoteroperably linked to a hairpin RNA (Lu et al., Nucleic Acids Res. Dec. 2,2004; 32(21):e171). The hairpin RNA may comprise a 5′ sequence ofroughly 19-24 nucleotides of sense strand target gene sequence followedby a spacer nucleotide of about 8-10 nucleotides followed by a sequenceof roughly 19-24 nucleotides of antisense sequence that is capable ofbase pairing with the preceding sense strand sequence. However, hairpinRNA-expressing plant transgenes containing sense/anti-sense arms rangingfrom 98 to 853 nucleotides can also be used (Wesley et al., Plant J.2001, 27(6):581-90). Vectors and methods for transgene-mediatedexpression of hairpin RNAs are disclosed in U.S. Patent Application Nos.20050164394, 20050160490, and 20040231016.

A first group of insect inhibitory agents that can be used incombination with a TIC807 protein for insect resistance management orexpanded insect inhibitory spectrum comprise insect inhibitory proteinsother than TIC807. A wide variety of insect inhibitory proteins derivedfrom B. thuringiensis, Photorhabdus sp., and/or Xenorhabdus sp. can beused.

For the control of sucking piercing insects such as Lygus, severalnon-TIC807 insect inhibitory proteins can be combined with TIC807expression in planta for greater control and/or resistance management.Such molecules expressed inplanta along with TIC807 may include ET29,ET37, TIC809, TIC810, TIC812, TIC127, TIC128 (PCT US 2006/033867),AXMI-027, AXMI-036, and AXMI-038 (WO 06/107761), AXMI-018, AXMI-020, andAXMI-021 (WO 06/083891), AXMI-010 (WO 05/038032), AXMI-003 (WO05/021585), AXMI-008 (US 2004/0250311), AXMI-006 (US 2004/0216186),AXMI-007 (US 2004/0210965), AXMI-009 (US 2004/0210964), AXMI-014 (US2004/0197917), AXMI-004 (US 2004/0197916), AXMI-028 and AXMI-029 (WO06/119457) and AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 andAXMI-004 (WO 04/074462). Presenting the combination of the inhibitoryprotein molecules, TIC809 (presented as SEQ ID NO: 10) and TIC810(presented as SEQ ID NO: 12) has been previously shown to be inhibitoryto the Western Tarnished Plant Bug (WTPB), Lygus hesperus Knight inbioassay (PCT US 2006/033867). The fusion proteins of TIC809 and TIC810,TIC 127 (presented as SEQ ID NO:14) and TIC 128 (presented as SEQ IDNO:16) may also be active against Lygus. The polynucleotide encodingTIC127 is comprised of the nucleic acid molecule encoding TIC809 linkedto the nucleic acid molecule encoding TIC810 by a polylinker nucleotidesequence (presented as SEQ ID NO:17) encoding the amino acid linkerpresented as SEQ ID NO:18. The polynucleotide encoding TIC128 iscomprised of the nucleic acid molecule encoding TIC810 linked to thenucleic acid molecule encoding TIC809 by a polylinker nucleotidesequence (presented as SEQ ID NO:17) encoding the amino acid linkerpresented as SEQ ID NO:18. Expression of TIC807 in combination withTIC127 or TIC128 may provide enhanced control of Lygus. Dicot plantssuch as cotton could be transformed with plant expression constructscontaining dicot-optimized nucleotide sequences encoding TIC807(presented as SEQ ID NO:6) along with TIC809 (presented as SEQ ID NO:9)and TIC810 (presented as SEQ ID NO:11), or TIC127 (presented as SEQ IDNO:13), or TIC128 (presented as SEQ ID NO:15) to provide enhancedresistance to Lygus or inhibition of additional species contained withinthe genus, Lygus.

For control of Lepidopteran pests, combinations of TIC807 proteins withLepidopteran-active proteins such as Cry1A proteins (U.S. Pat. No.5,880,275), Cry1B (U.S. patent application Ser. No. 10/525,318), Cry1C(U.S. Pat. No. 6,033,874), Cry1F, Cry1A/F chimeras (U.S. Pat. Nos.7,070,982; 6,962,705; and 6,713,063), and a Cry2Ab protein (U.S. Pat.No. 7,064,249) are specifically contemplated.

DNA sequences encoding TIC807 protein molecules and other insectinhibitory agents such as double stranded RNA molecules and/ornon-TIC807 proteins can be combined in a single plant either throughdirect transformation, by breeding, or a combination thereof. Multipletranscription units comprising a promoter and an insect inhibitory agentencoding region can be introduced on the same plant transformationvector or on different plant transformation vectors. When the two insectinhibitory agents are proteins, the coding regions for each may beseparated by a protease sensitive linker or even a self-processingprotease cleavage site (see U.S. Pat. No. 5,846,767). When the insectinhibitory agents are each introduced into distinct transgenic plants,those plants may be crossed to obtain a plant containing all of theinsect inhibitory agent encoding transgenes.

It is further anticipated that the combination of TIC807 proteinmolecules and other insect inhibitory agents such as double stranded RNAmolecules and/or non-TIC807 proteins can result in unexpectedsynergistic insect inhibitory effects that are not observed with eitherthe TIC807 insecticidal protein alone, the insect inhibitoryribonucleotide alone, or the non-TIC807 insect inhibitory protein alone.Synergistic effects include but are not limited to: i) quantitativechanges in LC₅₀ EC₅₀, IC₅₀, percent mortality, or percent stuntingvalues and ii) qualitative changes in the spectrum of insect inhibition(i.e., Hemipteran, Homopteran, and Lepidopteran insects inhibition) thatdoes not reflect the simple combination of the spectrum exhibited byeach insect inhibitory agent alone (i.e., the combination of Hemipteraninsect inhibition provided by one agent and Lepidopteran insectinhibition provided by another agent). A non-limiting example of aquantitative synergistic effect is a decrease in any LC₅₀, EC₅₀, and/orIC₅₀, value or an increase in percent mortality, or percent stuntingvalues observed in a combination that is more than additive. Anon-limiting example of a qualitative synergistic effect is control ofan insect pest with the combination of insect agents that is notobserved with either member alone. In this instance, the new insect pestcontrolled by the combination may be an insect pest within an order ofinsects (i.e., Hemipterans) where the insect inhibitory agents onlyinhibit other insect pests within that order of insects when used alone.

XIII. Isolated TIC807 Proteins and Biological Equivalents

Isolated TIC807 proteins are also provided herein. In one embodiment,the TIC807 proteins comprise proteins of at least 250 amino acids thathave at least 70% sequence identity to SEQ ID NO:5 and display insectinhibitory activity. The biologically functional equivalent peptides,polypeptides, and proteins contemplated herein should possess about 70%or greater sequence identity, preferably about 85% or greater sequenceidentity, and most preferably about 90% to 95% or greater sequenceidentity, to the sequence of, or corresponding moiety within, the TIC807polypeptide sequence. In certain embodiments of the invention,biologically functional equivalent peptides, polypeptides, and proteinspossessing about 80% or greater sequence identity, preferably about 85%,86%, 87%, 88%, 89% or greater sequence identity, and most preferablyabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greatersequence identity, to the sequence of TIC807 (SEQ ID NO:5)

Peptides, polypeptides, and proteins biologically functionallyequivalent to TIC807 include, but are not limited to, amino acidsequences containing conservative amino acid substitutions in the TIC807protein sequences. An example of TIC807 proteins that can be substitutedto obtain biological equivalents include, but are not limited to, theTIC807 protein sequence (SEQ ID NO:5). In such amino acid sequences, oneor more amino acids in the sequence is (are) substituted with anotheramino acid(s), the charge and polarity of which is similar to that ofthe native amino acid, i.e., a conservative amino acid substitution,resulting in a silent change.

Substitutes for an amino acid within the TIC807 polypeptide sequence canbe selected from other members of the class to which the naturallyoccurring amino acid belongs. Amino acids can be divided into thefollowing four groups: (1) acidic amino acids; (2) basic amino acids;(3) neutral polar amino acids; and (4) neutral non-polar amino acids.Representative amino acids within these various groups include, but arenot limited to: (1) acidic (negatively charged) amino acids such asaspartic acid and glutamic acid; (2) basic (positively charged) aminoacids such as arginine, histidine, and lysine; (3) neutral polar aminoacids such as glycine, serine, threonine, cysteine, cystine, tyrosine,asparagine, and glutamine; (4) neutral nonpolar (hydrophobic) aminoacids such as alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, and methionine.

Conservative amino acid changes within the TIC807 polypeptide sequencecan be made by substituting one amino acid within one of these groupswith another amino acid within the same group. Biologically functionalequivalents of TIC807 can have 10 or fewer conservative amino acidchanges, more preferably seven or fewer conservative amino acid changes,and most preferably five or fewer conservative amino acid changes. Theencoding nucleotide sequence (gene, plasmid DNA, cDNA, or synthetic DNA)will thus have corresponding base substitutions, permitting it to encodebiologically functional equivalent forms of TIC807.

As indicated, modification and changes may be made in the structure ofthe peptides of the present invention and DNA segments which encode themand still obtain a functional molecule that encodes a protein or peptidewith desirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second-generation molecule. In particular embodiments ofthe invention, mutated TIC807 proteins are contemplated to be useful forincreasing the insect inhibitory activity of the protein, andconsequently increasing the insect inhibitory activity and/or expressionof the recombinant transgene in a plant cell. The amino acid changes maybe achieved by changing the codons of the DNA sequence, according to thecodons given in Table 1.

TABLE 1 Amino Acid Amino Acids Codes Codons Alanine Ala (A) GCA GCC GCGGCU Cysteine Cys (C) UGC UGU Aspartic acid Asp (D) GAC GAU Glutamic acidGlu (E) GAA GAG Phenylalanine Phe (F) UUC UUU Glycine Gly (G) GGA GGCGGG GGU Histidine His (H) CAC CAU Isoleucine Ile (I) AUA AUC AUU LysineLys (K) AAA AAG Leucine Leu (L) UUA UUG CUA CUC CUG CUU Methionine Met(M) AUG Asparagine Asn (N) AAC AAU Proline Pro (P) CCA CCC CCG CCUGlutamine Gln (Q) CAA CAG Arginine Arg (R) AGA AGG CGA CGC CGG CGUSerine Ser (S) AGC AGU UCA UCC UCG UCU Threonine Thr (T) ACA ACC ACG ACUValine Val (V) GUA GUC GUG GUU Tryptophan Trp (W) UGG Tyrosine Tyr (Y)UAC UAU

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of biochemical orbiological activity. Since it is the interactive capacity and nature ofa protein that defines that protein's biological functional activity,certain amino acid sequence substitutions can be made in a proteinsequence, and, of course, its underlying DNA coding sequence, andnevertheless obtain a protein with like properties. It is thuscontemplated by the inventors that various changes may be made in thepeptide sequences of the disclosed compositions, or corresponding DNAsequences which encode said peptides without appreciable loss of theirbiological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, J Mol Biol. 157(1): 105-32,1982). It is accepted that the relative hydropathic character of theamino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis ofits hydrophobicity and charge characteristics (Kyte and Doolittle,Ibid). These are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within .+2is preferred, those which are within +1 are particularly preferred, andthose within +0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, states that the greatest local average hydrophilicity ofa protein, as governed by the hydrophilicity of its adjacent aminoacids, correlates with a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0.+0.1); glutamate (+3.0.+0.1); serine(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine(−0.4); proline (−0.5.+0.1); alanine (−0.5); histidine (−0.5); cysteine(−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine(−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

Non-Conservative Substitutions in the TIC807 Polypeptides

It is further recognized that non-conservative substitutions in TIC807polypeptide sequences can be made to obtain TIC807 polypeptides that arethe functional biological equivalents of the TIC807 polypeptidesdisclosed herein. In these instances, the non-conservative substitutionscan simply be tested for inhibition of fungal growth to identifynon-conservative substitutions that provide for functional biologicalequivalents of a given TIC807 polypeptide.

Fragments and Variants of TIC807

While the insect inhibitory polypeptide of the present inventionpreferably comprise a TIC807 protein sequence, fragments and variants ofthis sequence possessing the same or similar insect inhibitory activityas that of this insect inhibitory protein are also encompassed by thepresent invention. Thus contiguous sequences of at least 250 or moreamino acids in an TIC807 protein with insect inhibitory activity areanticipated by this invention. Fragments or variants of TIC807 withinsect inhibitory activity that are anticipated by this invention canalso comprise amino acid substitutions, deletions, insertions oradditions in an TIC807 protein sequence.

The insect inhibitory polypeptide of the present invention preferablycomprises the TIC807 protein sequence (SEQ ID NO:5), fragments andvariants of this sequence possessing the same or similar insectinhibitory activity as that of this particular TIC807 protein are alsoencompassed by the present invention are anticipated by this invention.Thus contiguous sequences of at least 250 or more amino acids in SEQ IDNO:5 with insect inhibitory activity are anticipated by this invention.The insect inhibitory TIC807 fragments can also comprise fragments withat least 260, at least 270, at least 280, at least 290, or at least 300amino acid residues of the 309 amino acid TIC807 sequence of SEQ IDNO:5. The fragments or variants with insect inhibitory activity that areanticipated by this invention can also comprise amino acidsubstitutions, deletions, insertions or additions of the sequence shownin SEQ ID NO:5.

Fragments of the mature TIC807 protein can be truncated forms whereinone or more amino acids are deleted from the N-terminal end, C-terminalend, the middle of the protein, or combinations thereof with insectinhibitory activity are also anticipated by this invention. Thesefragments can be naturally occurring or synthetic mutants of TIC807, andretain the insect inhibitory activity of TIC807. A preferred TIC807protein that can be used to obtain truncated derivatives with insectinhibitory activity is the TIC807 protein of SEQ ID NO:5.

Variants of TIC807 include forms wherein one or more amino acids has(have) been inserted into the natural sequence. These variants can alsobe naturally occurring or synthetic mutants of TIC807, and retain theinsect inhibitory activity of TIC807.

Combinations of the foregoing, i.e., forms of the insect inhibitorypolypeptide containing both amino acid deletions and additions, are alsoencompassed by the present invention. Amino acid substitutions can alsobe present therein as well.

The fragments and variants of TIC807 encompassed by the presentinvention should preferably possess about 70-75% or greater sequenceidentity, more preferably about 80%, 85%, 88% or greater sequenceidentity, and most preferably about 90% to 95% or greater amino acidsequence identity, to the corresponding regions of the mature TIC807protein having the corresponding amino acid sequences shown in SEQ IDNO:5.

Use of Structure Function Relationships to Design Insect InhibitoryTIC807 Variants

This invention also contemplates the use of structure functionrelationships to design additional insect inhibitory TIC807 proteinvariants. It is first contemplated that a structure could be obtained bycrystallographic analysis of TIC807 crystals. Such structures areanticipated to reveal domains of the TIC807 protein involved in insectreceptor binding, pore formation in the insect gut, multimerization withTIC807, protease sensitivity and/or protease resistance that contributeto the insect inhibitory activity of TIC807.

It is further anticipated that comparisons between TIC807 and otherrelated proteins may permit extrapolation of protein domains thatcontribute to the insecticidal activity of TIC807 proteins. In thisregard, it is noted that TIC807 has some similarity to a family ofMTX-like proteins. This Mtx-like family of proteins is named after theBacillus sphericus proteins Mtx2 (Thanabalu and Porter, Gene. 170(1):85,1996; NCBI Accession No. 2211294A) and Mtx3 (Liu et al., Appl EnvironMicrobiol. 62(6):2174, 1996; NCBI Accession No. AAB36661) and includesCry15Aa (SEQ ID NO:41), Cry33Aa (NCBI Accession No. AAL26871), Cry23Aa(NCBI Accession No. AAF76375), Cry38Aa (NCBI Accession No. AAK64559),CryC35 (NCBI Accession No. CAA63374), the 40KD protein (NCBI AccessionNo. AAA22332), and CryNT32 (NCBI Accession No. AAL26870). It is alsobelieved that TIC807 is distantly related to the aerolysin family ofproteins that include cryET33 (WO 97/17600), and TIC901 (U.S. PatentApplication No. 20060191034). Aerolysins are a group of proteins thatmultimerize and form pores in membranes and are known toxins (Parker etal., Mol. Microbiol. 19(2):205, 1996). In particular, crystallographicstructure determinations indicate that beta-sheet domains of aerolysinsare involved in forming membrane pores (Rossjohn et al., J Struct Biol.121(2):92, 1998). Domains of TIC807 proteins could be swapped withsimilar domains from other MTX-like or Aerolysin family proteins toidentify domains involved in insect receptor binding, pore formation inthe insect gut, multimerization with TIC807, protease sensitivity and/orprotease resistance that contribute to the insect inhibitory activity ofTIC807. Data from the domain swapping experiments can be compared andotherwise extrapolated to structural data for Mtx-like protein familymembers to elucidate domains that provide for different insecticidalactivities, improved insecticidal activities, improved bindingcharacteristics, improved pore forming capabilities.

Having identified certain protein domains of the TIC807 proteins thatprovide for insect inhibitory properties of the TIC807 protein (i.e.,insect receptor binding, pore formation in the insect gut,multimerization with TIC807, protease sensitivity and/or proteaseresistance), it is further anticipated that these regions can be moreextensively mutagenized. Once mutagenized, variant TIC807 proteins canbe subjected to either biochemical (i.e., insect receptor binding, poreformation in the insect gut, multimerization with TIC807, proteasesensitivity and/or protease resistance) or biological assays (i.e.,insect inhibition assays) to identify those variants that conferimproved biochemical and/or insect inhibitory activities. Additionaliterative rounds of mutagenesis and assay of those identified variantsis also contemplated. Various procedures for the molecular evolution ofisolated proteins that are either known to those skilled in the art(Stemmer, W., Proc. Natl. Acad. Sci. USA 91: 10747, 1994; Yuan et al.,Microbiol. Mol. Biol. Rev. 69(3):373, 2005) or are provided by otherentirely distinct methods can be employed to generate the TIC807 proteinvariants.

Isolated TIC807 Proteins of at Least 9 Amino Acids

In other embodiments of this invention, isolated proteins that comprisea polypeptide sequence of at least 9 amino acids in length that iscontained within SEQ ID NO:5 are provided. At least two distinct usesfor TIC807 peptide sequences of at least 9 amino acids are contemplated.

First, it is contemplated that TIC807 peptide sequences of at least 9amino acids can be substituted into distinct protein sequences to conferall or a subset of the insect inhibitory activities of a TIC807 proteinon the resultant TIC807-peptide substituted protein. Insect inhibitoryactivities conferred by the TIC807 peptide sequences can compriseinhibition of a hemipteran pest including, but not limited to, Lygus.Without being limited by theory, it is believed that TIC807 peptidesequences of at least 9 amino acids can provide: 1) improved crystalformation, 2) improved protein stability or reduced proteasedegradation, 3) improved insect membrane receptor recognition andbinding, 4) improved oligomerization or channel formation in the insectmidgut endothelium, and 5) improved insecticidal activity orinsecticidal specificity due to any or all of the reasons stated abovewhen inserted into another protein. Larger TIC807 peptide sequences ofat least 12, at least 16, at least 32, at least 50 or at least 100 aminoacid residues from SEQ ID NO:5 can also be substituted into distinctprotein sequences to obtain insect inhibitory TIC807-peptide substitutedproteins.

TIC807-peptide substituted protein can be synthesized by techniquesincluding, but not limited to, site-specific mutagenesis (Kunkel, T. A.et al. Meth. Enzymol. 154: 367, 1987), DNA shuffling (Stemmer, W., Proc.Natl. Acad. Sci. USA 91: 10747, 1994), PCR.™. overlap extension (Hortonet al., Gene 77: 61, 1989), any of the protein molecular evolutionmethods (Yuan et al., Microbiol. Mol. Biol. Rev. 69(3):373, 2005),direct synthesis, combinations of these methods, or by other entirelydistinct methods that provide for TIC807-peptide substituted proteins.In particular, TIC807-substituted proteins derived by insertion orsubstitution of TIC807 peptide sequences of at least 9 amino acids intoinsect inhibitory proteins derived from Bacillus thuringiensis arecontemplated. Exemplary Bacillus thuringiensis proteins that can besubstituted with TIC807 polypeptides to obtain TIC807-substitutedproteins with insect inhibitory activity include, but are not limitedto, Cry15Aa1 (Brown & Whiteley, 1992, J Bacteriol 174 549-557; SEQ IDNO:41), CryET29 (U.S. Pat. No. 6,093,695), Cyt1Ba1 (U.S. Pat. No.5,723,440), Bacillus thuringiensis israelensis Cyt toxins (U.S. Pat. No.5,885,963), and distinct Lygus active Bacillus thuringiensis crystalproteins AXMI-027, AXMI-036 and AXMI-038 disclosed in U.S. PatentApplication Publication No. 20060242732. Other proteins that can besubstituted with TIC807 polypeptides to obtain TIC807-substitutedproteins with insect inhibitory activity include, but are not limitedto, the Mtx2 (Thanabalu and Porter, Gene. 170(1):85, 1996; NCBIAccession No. 2211294A), Mtx3 (Liu et al., Appl Environ Microbiol.62(6):2174, 1996; NCBI Accession No. AAB36661), Cry15Aa (SEQ ID NO:41),Cry33Aa (NCBI Accession No. AAL26871), Cry23Aa (NCBI Accession No.AAF76375), Cry38Aa (NCBI Accession No. AAK64559), CryC35 (NCBI AccessionNo. CAA63374), the 40KD protein (NCBI Accession No. AAA22332), CryNT32(NCBI Accession No. AAL26870), cryET33 (WO 97/17600), and TIC901 (U.S.Patent Application Publication No. 20060191034).

It is also contemplated that isolated TIC807 proteins of between about250 and about 309 amino acids can also be used for antibody productionor insect inhibition. These isolated TIC807 polypeptide sequences of theinvention have at least about 70%, at least about 90%, at least about95% or 100% sequence identity to a corresponding polypeptide sequencecontained within SEQ ID NO:5. These TIC807 proteins can further comprisea covalently linked indicator reagent, an amino acid spacer, an aminoacid linker, a signal sequence, a chloroplast transit peptide sequence,a vacuolar targeting sequence, or a stop transfer sequence.

It is also contemplated that isolated TIC807 peptide sequences of atleast 9 contiguous amino acids of SEQ ID NO:5 can be used as immunogensor epitopes to prepare antibodies that recognize TIC807 proteins. Suchantibodies are useful for detecting TIC807 proteins in transgenicplants, in commodity products derived from transgenic plants, inmicroorganisms or in recombinant DNA expression libraries that containcloned TIC807 sequences. The TIC807 polypeptides can be at least 9, atleast 12, at least 16, or at least 32 amino acids in length. When theTIC807 peptide sequence is at least 32 amino acids in length it has atleast about 80%, 90%, or 95% sequence identity to a correspondingpolypeptide sequence contained within SEQ ID NO:5. The peptides can belinked to a carrier protein such as KLH or albumin to facilitateantibody production.

The identification of TIC807 protein immunodominant epitopes, and/ortheir functional equivalents, suitable for use in vaccines is arelatively straightforward matter. For example, one may employ themethods of Hopp, as taught in U.S. Pat. No. 4,554,101, which teaches theidentification and preparation of epitopes from amino acid sequences onthe basis of hydrophilicity. The methods described in several otherpapers, and software programs based thereon, can also be used toidentify epitopic core sequences (see, for example, U.S. Pat. No.4,554,101). The amino acid sequence of these “epitopic core sequences”may then be readily incorporated into peptides, either through theapplication of peptide synthesis or recombinant DNA technology.

Preferred TIC807 peptides for use in accordance with the presentinvention will generally be on the order of about 9 to about 20 aminoacids in length, and more preferably about 9 to about 15 amino acids inlength. It is proposed that shorter antigenic TIC807 protein-derivedpeptides will provide advantages in certain circumstances, for example,in the preparation of immunologic detection assays. Exemplary advantagesinclude the ease of preparation and purification, the relatively lowcost and improved reproducibility of production, and advantageousbiodistribution.

It is proposed that particular advantages of the present invention maybe realized through the preparation of synthetic peptides which includemodified and/or extended epitopic/immunogenic core sequences whichresult in a “universal” epitopic peptide directed to TIC807 proteins,and in particular to TIC807-related sequences. These epitopic coresequences are identified herein in particular aspects as hydrophilicregions of the particular polypeptide antigen. It is proposed that theseregions represent those which are most likely to promote T-cell orB-cell stimulation, and, hence, elicit specific antibody production.

An epitopic core sequence, as used herein, is a relatively short stretchof amino acids that is “complementary” to, and therefore will bind,antigen binding sites on the TIC807 protein-directed antibodiesdisclosed herein. Additionally or alternatively, an epitopic coresequence is one that will elicit antibodies that are cross-reactive withantibodies directed against the peptide compositions of the presentinvention. Thus, certain epitope core sequences of the present inventionmay be operationally defined in terms of their ability to compete withor perhaps displace the binding of the desired protein antigen with thecorresponding protein-directed antisera.

In general, the size of the polypeptide antigen is not believed to beparticularly crucial, so long as it is at least large enough to carrythe identified core sequence or sequences. The smallest useful coresequence anticipated by the present disclosure would generally be on theorder of about 9 amino acids in length, with sequences on the order of10 to 20 being more preferred. Thus, this size will generally correspondto the smallest peptide antigens prepared in accordance with theinvention. However, the size of the antigen may be larger where desired,so long as it contains a basic epitopic core sequence.

XIV. TIC807 Antibody Compositions and Methods of Making Antibodies

In particular embodiments, the inventors contemplate the use ofantibodies, either monoclonal or polyclonal which bind to the TIC807proteins disclosed herein. Means for preparing and characterizingantibodies are well known in the art (see, e.g., Using Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, 1999). The methods forgenerating monoclonal antibodies (mAbs) generally begin along the samelines as those for preparing polyclonal antibodies. Briefly, apolyclonal antibody is prepared by immunizing an animal with animmunogenic composition in accordance with the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically the animalused for production of antisera is a rabbit, a mouse, a rat, a hamster,a guinea pig or a goat. Because of the relatively large blood volume ofrabbits, a rabbit is a preferred choice for production of polyclonalantibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or protein immunogen toa carrier. Exemplary and preferred carriers are keyhole limpethemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such asovalbumin, mouse serum albumin or rabbit serum albumin can also be usedas carriers. Means for conjugating a peptide, polypeptide, or protein toa carrier protein are well known in the art and include usingglutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester,carbodiimide and bis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

Monoclonal antibodies (mAbs) may be readily prepared through use ofwell-known techniques, such as those exemplified in U.S. Pat. No.4,196,265. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified antifungal protein, polypeptide or peptide. Theimmunizing composition is administered in a manner effective tostimulate antibody producing cells. Rodents such as mice and rats arepreferred animals, however, the use of rabbit, sheep, or frog cells isalso possible. The use of rats may provide certain advantages but miceare preferred, with the BALB/c mouse being most preferred as this ismost routinely used and generally gives a higher percentage of stablefusions.

Also contemplated are methods of genetic immunization to obtain eithermonoclonal or polyclonal antibodies which bind to the TIC807 proteinsdisclosed herein. In these methods, the gene encoding the TIC807 proteinis operably linked to a promoter that is active in mammalian cells.Isolated plasmid DNA comprising the mammalian cell expression cassettecomprising the TIC807 encoding protein is then directly injected intothe animal to elicit an immune response to the encoded TIC807 protein.Animals that can be used as injection hosts for genetic immunizationinclude, but are not limited to, mice, rats, rabbits, goats, cows, orhorses. Although a variety of injection regimens can be used, oneexemplary regimen would comprise injection of plasmid DNA dissolved inphosphate-buffered saline or other suitable buffer at a concentration ofapproximately 1-2 mg plasmid DNA/ml and at a dose of about 100ug/injection/animal (i.e., for a mouse, rat or rabbit). About 3-4injections can be made in each animal in two week intervals. Geneticimmunization is described in Chambers and Johnston, Nature Biotechnol.(21): 1088, 2003). Contract research organizations also conduct geneticimmunization experiments to obtain antibodies (QED Bioscience Inc., SanDiego, Calif., USA).

Examples of useful mammalian expression cassettes that can be used forgenetic immunization include, but are not limited to, the pcDNA3.1vector (Invitrogen, Carlsbad, Calif., USA) that provides a CMV promoterfor expression of operably linked genes or the pRc/RSV vector(Invitrogen, Carlsbad, Calif., USA). In cases where high levels ofantigen expression is cytotoxic, a weaker promoter, such as the SV40promoter, can be used to express the antigen. It is anticipated thateither the native TIC807 gene (SEQ ID NO:4) or the synthetic TIC807 gene(SEQ ID NO:6) can be operably linked to promoters and polyadenylationelements that are active in mammalian cells to obtain plasmids suitablefor genetic immunization. However, the design and synthesis of otherTIC807 encoding sequences for expression in mammalian hosts bybacktranslation of the TIC807 amino acid sequence (SEQ ID NO:5) is alsocontemplated. Mammalian expression vectors that further comprise signalpeptide sequences that provide for extracellular secretion and/ortransmembrane insertion of operably linked sequences encoding TIC807proteins are also contemplated.

XV. TIC807 Protein Screening and Detection Kits

The present invention contemplates methods and kits for screeningsamples suspected of containing TIC807 proteins or TIC807protein-related polypeptides, or cells producing such polypeptides. Inthe particular embodiments contemplated herein, the methods and kitsdetect the TIC807 protein. A kit may contain one or more antibodies ofthe present invention, and may also contain reagent(s) for detecting aninteraction between a sample and an antibody of the present invention.The provided reagent(s) can be radio-, spectrophotometrically-,fluorescently- or enzymatically-labeled. The provided reagents mayinclude a substrate that is converted to a product that can be detectedby spectrophotometry, luminometry, or fluorescence. The kit can containa known radiolabeled or hapten-labeled agent capable of binding orinteracting with an antibody of the present invention.

The reagent(s) of the kit may be provided as a liquid solution, attachedto a solid support or as a dried powder. Preferably, when the reagent(s)are provided in a liquid solution, the liquid solution is an aqueoussolution. Preferably, when the reagent(s) provided are attached to asolid support, the solid support can be chromatograph media, a testplate having a plurality of wells, or a microscope slide. When thereagent(s) provided are a dry powder, the powder can be reconstituted bythe addition of a suitable solvent, that may be provided.

In still further embodiments, the present invention concernsimmunodetection methods and associated kits. It is proposed that theTIC807 proteins or peptides of the present invention may be employed todetect antibodies having reactivity therewith, or, alternatively,antibodies prepared in accordance with the present invention, may beemployed to detect TIC807 proteins or TIC807 protein-relatedepitope-containing peptides. In general, these methods will includefirst obtaining a sample suspected of containing such a protein, peptideor antibody, contacting the sample with an antibody or peptide inaccordance with the present invention, as the case may be, underconditions effective to allow the formation of an immunocomplex, andthen detecting the presence of the immunocomplex.

In general, the detection of immunocomplex formation is quite well knownin the art and may be achieved through the application of numerousapproaches. For example, the present invention contemplates theapplication of ELISA, RIA, immunoblot (e.g., dot blot), indirectimmunofluorescence techniques and the like. Generally, immunocomplexformation will be detected through the use of a label, such as aradiolabel or an enzyme tag (such as alkaline phosphatase, horseradishperoxidase, or the like). Of course, one may find additional advantagesthrough the use of a secondary binding ligand such as a second antibodyor a biotin/avidin ligand binding arrangement, as is known in the art.

For assaying purposes, it is proposed that virtually any samplesuspected of comprising either a TIC807 protein or peptide or a TIC807protein-related peptide or antibody sought to be detected, as the casemay be, may be employed. It is contemplated that such embodiments mayhave application in the titering of antigen or antibody samples, in theselection of hybridomas, and the like. In related embodiments, thepresent invention contemplates the preparation of kits that may beemployed to detect the presence of TIC807 proteins or related peptidesand/or antibodies in a sample. Samples may include cells, cellsupernatants, cell suspensions, cell extracts, enzyme fractions, proteinextracts, or other cell-free compositions suspected of containing TIC807proteins or peptides. Generally speaking, kits in accordance with thepresent invention will include a suitable TIC807 protein, peptide or anantibody directed against such a protein or peptide, together with animmunodetection reagent for detecting antibody/antigen complexes,instructions for the use of these materials, and a means for containingthe antibody or antigen and reagent. The immunodetection reagent willtypically comprise a label associated with the antibody or antigen, orassociated with a secondary binding ligand. Exemplary ligands mightinclude a secondary antibody directed against the first antibody orantigen or a biotin or avidin (or streptavidin) ligand having anassociated label. Of course, as noted above, a number of exemplarylabels are known in the art and all such labels may be employed inconnection with the present invention.

The container will generally include a vial into which the antibody,antigen or detection reagent may be placed, and preferably suitablyaliquotted. The kits of the present invention will also typicallyinclude a means for containing the antibody, antigen, and reagentcontainers in close confinement for commercial sale. Such containers mayinclude injection or blow-molded plastic containers into which thedesired vials are retained.

In view of the foregoing, it will be seen that the several advantages ofthe invention are achieved and attained.

The embodiments were chosen and described in order to best explain theprinciples of the invention and its practical application to therebyenable others skilled in the art to best utilize the invention invarious embodiments and with various modifications as are suited to theparticular use contemplated.

EXAMPLES

The following disclosed embodiments are merely representative of theinvention, which may be embodied in various forms. Thus, specificstructural and functional details disclosed herein are not to beinterpreted as limiting.

Example 1 Identification of Bacillus thuringiensis Strain EG2934

This example describes Bacillus thuringiensis strain EG2934 and crystalproteins derived from this strain

Bacillus thuringiensis strains are well known for their ability toproduce parasporal crystals that contain proteins with diverseinsecticidal activities against Lepidopteran, Coleopteran, and Dipteraninsect species. These parasporal crystals exhibit a variety of geometricshapes when viewed by phase-contrast microscopy and have been describedas irregular, cuboidal, rod-shaped, rhomboidal, bipyramidal, et cetera.B. thuringiensis strains exhibiting Lepidopteran toxic activity appearto be more common than B. thuringiensis strains exhibiting toxicity toother insect species. Parasporal crystals exhibiting a bipyramidal shapeare frequently associated with Lepidopteran toxic B. thuringiensisisolates. This bipyramidal crystal structure-function relationship withLepidopteran activity provides an advantage when screeninguncharacterized B. thuringiensis strains, allowing a rapid selection ofstrains that may exhibit insecticidal activity directed to insects otherthan Lepidopteran species. Strain EG2934 was selected on this basis asit appeared when viewed by phase-contrast microscopy to contain welldefined crystals lacking a bipyramidal structure. In order to establishwhich crystal proteins produced by this strain possessed insecticidalactivity, the genes encoding these proteins were cloned and expressed ina well-characterized toxin-free and acrystalliferous B. thuringiensishost strain. Four crystal proteins, ranging in size from approximately35 kilodaltons (kDa) to approximately 120 kDa were identified in crystalpreparations produced by B. thuringiensis strain EG2934. As a matter ofroutine screening these proteins were submitted for testing againstknown plant insect pests for toxicity. One protein from B. thuringiensisstrain EG2934, designated as TIC807, was found to be toxic to thepiercing-sucking insects, Lygus hesperus and Lygus lineolaris.

Example 2 Characterization of Crystal Proteins Produced by the B.thuringiensis Strain EG2934

This example illustrates the characterization of crystal proteinsisolated from the B. thuringiensis strain EG2934 and the subsequentinitial characterization of the Lygus active toxin protein, TIC807.

B. thuringiensis strain EG2934 was grown at 25 to 28 degrees Celsius inC2 sporulation medium (Donovan et al., Mol. Gen. Gent. 214: 365-372,1988) for 3 to 4 days or until fully sporulated and lysed. Spores andcrystals were collected by centrifugation and resuspended in wash buffer(10 mM Tris-HCl, 0.1 mMr EDTA, 0.005 percent Triton X-100, pH 6.8) andcollected again by centrifugation. The spore-crystal pellets wereresuspended in wash buffer at one tenth the original culture volume.Crystal proteins in the 10× concentrates were analyzed by SDSpolyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrationswere determined by densitometry using bovine serum albumin (BSA) as astandard.

B. thuringiensis strain EG2934 produces crystal proteins ofapproximately 120, 110, 65 and 35 kilodaltons (kDA) upon sporulation.Proteins from EG2934 were resolved by SDS-PAGE. After electrophoresis,the proteins were transferred to a PVDF membrane (BioRad, Hercules,Calif.) following standard western blotting procedures. After transfer,the proteins bound to each membrane were subjected to N-terminalsequencing, using standard automated Edman degradation procedures. TheN-terminal amino acid sequence of TIC807 is presented as SEQ ID NO:1.Queries of available public databases failed to identify a significantmatch to this sequence, suggesting the TIC807 protein may be novel. Twodegenerate oligonucleotides primers, designated djc-pr12 (SEQ ID NO:2)and djc-pr113 (SEQ ID NO:3) were designed based upon the amino acidsequence, SEQ ID NO:1 to serve as hybridization probes for the isolationof a B. thuringiensis genes encoding the TIC807 protein and TIC807homologs.

Example 3 Isolation and Characterization of TIC807 Isolated from B.thuringiensis Strain EG2934

This example illustrates the screening for phage clones containing DNAencoding the TIC807 protein. The cloning and sequencing of the DNAencoding the TIC807 protein is also described. The method describedbelow can also be applied to recovery of DNA sequences encoding TIC807homologs and related genes in plasmid, cosmid or phage libraries derivedfrom other B. thuringiensis strains.

The oligonucleotide primers described in example 2, djc-pr12 anddjc-pr13 (SEQ ID NO:2 and SEQ ID NO:3, respectively), were used ashybridization probes to probe a library constructed using size selectedDNA isolated from B. thuringiensis strain EG2934. In the degenerateoligonucleotides of SEQ ID NO:2 (AAYGCDATHA AYTAYTGGGG DCCDAARAAY) andSEQ ID NO:3 (TGGGGDCCDA ARAAYAAYAA YGARATWCAR), Y residues represent amixture of C or T residues, D residues represent a mixture of A, G, or Tresidues, H residues represent a mixture of A, C, or T residues, Rresidues represent a mixture of A or G residues, and W residuesrepresent a mixture of A or T residues. Total DNA from B. thuringiensisstrain EG2934 was prepared using a protocol that employs a CTABextraction step (Current Protocols in Molecular Biology (1997) JohnWiley and Sons, Inc.). This DNA failed to cut with the restrictionenzyme Sau3AI, a 4-base cutter typically used to prepare genomiclibraries. Surprisingly, an isoschizimer of Sau3AI that is sensitive toDNA methylation, MboI, digested the genomic DNA efficiently. Thedigested DNA was size fractionated on a 1% agarose 1×TBE gel. MboIfragments of 7-12 kb were extracted from gel slices using standardprotocols and ligated into a BamHI-digested λ-Zap Express vector andpackaged into phage particles using a GigaPack III Gold kit (Stratagene,La Jolla, Calif.). After phage amplification, transfections were platedand plaque lifts performed with Duralon UV nylon membranes (Stratagene,La Jolla, Calif.). The membranes were incubated at 50 degrees Celsius ina standard prehybridization/hybridization buffer containing 5×SSC, 0.1%N-lauroyl sarcosine, 0.02% SDS, 1% Blocking Reagent (Roche,Indianapolis, Ind., Cat. No. 1585762), 0.1 mg/mL poly-A, and 4 ug/mLpoly-dA for several hours. The oligonucleotides were labelled on the 3′end with digoxigenin (DIG) using terminal transferase, DIG-11-dUTP (DIGoligonuceotide tailing kit; Roche, Indianapolis, Ind., Cat. No. 1 417231) and protocols recommended by the manufacturer. A 1:1 mixture of theDIG-labeled oligos djc-pr12 and djc-pr13 was added and the membranesincubated overnight at 50 degrees Celsius. Filters were washed twice for15 minutes at room temperature in 3×SSC, 0.1% SDS and twice for 15minutes at 50 degrees Celsius in 1×SSC, 0.1% SDS. Hybridizing plaqueswere visualized by chemiluminescence using the Roche (Indianapolis,Ind.) Wash and Block buffer set and protocols (Roche, Indianapolis,Ind., Cat. No. 11585762001), anti-DIG-alkaline phosphatase Fab fragments(Roche, Indianapolis, Ind., Cat. No. 11093274910) and the substrate CSPD(Disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2-(5-chloro)tricyclo[3.3.1.13.7]decan}-4-yl)phenyl phosphate) (Roche, Indianapolis, Ind.).Twelve recombinant clones were picked from the primary plates, diluted,plated, and re-probed with the DIG-labeled oligos. Only two rounds ofplating/probing were required to obtain pure clones. All twelve phageclones were subjected to excision reactions yielding 12 individualphagemid clones. These were digested with SalI and NotI to release thecloned inserts. The digests were resolved on a 1% agarose 1×TBE gel andblotted to a Nytran® membrane for Southern analysis. The insert sizeswere much smaller than expected, ranging in size from 2-5 kb.Nevertheless, the Southern analysis demonstrates the presence ofhybridizing DNAs in 9 of 12 clones.

Thermal amplification reactions using combinations of the degenerateoligos, djc-pr12 and djc-pr13 with primers specific for vector sequencesflanking the inserts were run to map the location of the TIC807 codingregion on the phagemid clones. Based upon these results, it wasdetermined that the entire gene was present on four different phagemidclones designated pIC17039 through pIC17042. The insert size and strainidentification corresponding to each of these phagemid clones is shownin Table 2.

TABLE 2 Escherichia coli strains containing TIC807 phagemids and theassociated insert size. Estimated Insert Strain Phagemid size(kilobases) SIC8087 pIC17039 2.3 SIC8088 pIC17040 3.5 SIC8089 pIC170415.5 SIC8090 pIC17042 1.5

The TIC807 gene insert on plasmid pIC17042 was sequenced. The deducedcoding region (presented as SEQ ID NO:4) encodes a protein of 309 aminoacids and is presented as SEQ ID NO:5. A BlastP (version 2.2.9) searchof the non-redundant protein database revealed the closest match to theTIC807 protein sequence was to the B. thuringiensis crystal proteinCry15Aa1 (SEQ ID NO:41). A Needleman-Wunsch global alignment of the twoproteins demonstrated a 25.5% sequence identity between the TIC807protein sequence and the protein sequence of Cry15Aa1. The alignment ofTIC807 (SEQ ID NO:5) and Cry15Aa (SEQ ID NO:41) is illustrated inFIG. 1. Although the TIC807 and Cry15Aa1 proteins are not highlyconserved, localized regions of the proteins display complete sequenceconservation over short stretches of contiguous amino acid sequences ofup to seven residues in length.

Escherichia coli strain SIC8088 harboring vector pIC17040 was depositedon Mar. 16, 2007 with the Agricultural Research Culture Collection,Northern Regional Research Laboratory (NRRL) in Peoria, Ill. and givenAccession No. NRRLB-50030.

Example 4 Expression of TIC807 in a Toxin-Free B. thuringiensis HostStrain

This example illustrates the cloning of the TIC807 coding region into aplasmid for expression in a toxin-free B. thuringiensis host strain.This example also illustrates a process by which a protein toxin such asTIC807 can be produced in purity for bioassay against insect pests.

The TIC807 plasmids pIC17040 and pIC17041 (Table 1) were digested withthe restriction endonucleases NotI and XmaI and electrophoresed on a 1%agarose gel in 1×TBE. The resulting single DNA fragments were purifiedfollowing agarose gel electrophoresis using a Qiagen (Valencia, Calif.)DNA purification kit following the manufacturer's protocol. Likewise,the E. coli-B. thuringiensis shuttle vector pEG854 (Baum et al. 1990)was digested with the restriction endonucleases NotI and XmaI and anapproximate 4.3 kilobase DNA fragment containing the Bt plasmidreplication origin ori43 and the chloramphenicol acetyltransferase (cat)gene was purified following agarose gel electrophoresis. Ligation of theTIC807 gene fragments with the ori43-cat gene fragment yielded plasmidscapable of replicating in B. thuringiensis. The ligation products wereused to transform the acrystalliferous (Cry-) B. thuringiensis hoststrain EG10650 to chloramphenicol resistance by electroporation,yielding the recombinant B. thuringiensis isolates SIC8091, SIC8092,SIC8093, and SIC8094 depicted in Table 3.

TABLE 3 B. thuringiensis strain containing plasmids for the expressionof TIC807 Estimated Insert Strain Plasmid size (kilobases) SIC8091pIC17043 3.5 SIC8092 pIC17044 3.5 SIC8093 pIC17045 5.5 SIC8094 pIC170465.5

The recombinant strains were grown at 25 to 28 degrees Celsius in C2medium for 3-4 days or until fully sporulated and lysed. Spores andcrystals were collected by centrifugation (e.g., 4000×g for 30 minutes),resuspended in wash buffer (10 mM Tris-HCl, 0.1 mM EDTA, 0.005% TritonX-100, pH 6.8), and collected again by centrifugation. The spore-crystalpellets were resuspended in wash buffer at 1/10 th the original culturevolume. Crystal proteins present in these 10× C2 concentrates wereanalyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE). All fourrecombinant strains produced a crystal protein of the expected apparentmolecular mass of approximately 35 kDa. Protein concentrations weredetermined by densitometry using bovine serum albumin (BSA) as astandard.

Example 5 TIC807 is Toxic to Lygus hesperus and Lygus lineolaris

This example illustrates the feeding assay used to identify the TIC807protein molecule as being toxic to the western tarnished plant bug(WTPB), Lygus hesperus and the tarnished plant bug (TPB), Lyguslineolaris. The WTPB and TPB are phytophagous, piercing-sucking insectsthat attack numerous weeds and crops. The WTPB and TPB damageagricultural crops, including cotton, by direct feeding damage. Becausethe WTPB and TPB feed by piercing-sucking, the assay used to testprotein toxins for this class of insects must allow for the insect'snatural feeding behavior. The feeding assay employed was based on a 96well format and a sachet system as described by Habibi et al., (Archivesof Insect Biochem. and Phys. 50: 62-74 (2002)). The artificial diet wassupplied by Bio-Serv® (Bio-Serv® Diet F9644B, Frenchtown, N.J.), thecomponents of which are presented in Table 4.

TABLE 4 The Bio-Serv ® F9644B WTPB artificial diet. Diet IngredientsGrams/Liter Wheat Germ, Stabilized 44.60 Cholesterol 0.50 RNA 5.00Vitamin mix, Vanderzant 8.90 Para-Aminobenzoic acid 0.18 Niacin 0.18Vitamin E Acetate 0.10 Aureomycin 0.10 Streptomycin Sulfate 0.135Carageenan (Irish Moss) 3.00 Lima Beans, ground 45.00 CaseinHydrolaysate 17.90 Salt Mix, Hesperus 2.90 Sucrose 27.10 Lecithin,Liquid, Soy 0.50 Safflower Oil 0.20 Chicken eggs (4) Not added

Five hundred and eighteen milliliters of autoclaved, boiling water werecombined with 156.3 grams of Bio-Serv® Diet F9644B in a surfacesterilized blender. Four surface sterilized chicken eggs were broken andthe contents were added to the blender containing the diet mix. Themixture was blended until smooth and adjusted to one liter of volume andallowed to cool. Toxin samples were prepared by mixing the TIC807 toxinprotein preparation in the desired concentration with an equivalentvolume of the blended diet.

A sheet of Parafilm® (Pechiney Plastic Packing, Chicago, Ill.) wasplaced over a 96-well format vacuum manifold (Analytical ResearchSystems, Gainesville, Fla.) with a vacuum of approximately −20millimeters mercury, which is sufficient to cause extrusion of theParafilm® into the wells. Forty microliters of test sample were added tothe Parafilm® wells. A sheet of Mylar film (Clear Lam Packaging, Inc.,Elk Grove Village, Ill.) was then placed over the Parafilm® and sealedgently with a tacking iron (Bienfang Sealector II, Hunt Corporation,Philadelphia, Pa.). The Parafilm® sachets were then placed over aflat-bottom 96-well plate containing the Lygus eggs suspended inagarose. Upon hatching, Lygus nymphs will feed by piercing the sachetthat is presented above them. Without being limited by theory, it isbelieved that extraoral digestion in the sachet may lead to proteolysisand degradation prior to ingestion by the insect. To assure intactprotein was being presented to the insect in its diet, the diet sachetswere replaced every two days. This enhancement in theory allows forlonger presentation of the intact toxin proteins in the insect diet overthe course of the feeding assay. In addition, lower concentrations ofputative toxin protein can be tested since greater amounts of proteinwill not be required to compensate for potential extraoral digestiveeffects. Insect diet sachets were replaced on days two and four.Stunting and mortality scores were determined on day 5 and compared tothe untreated check (UTC).

Tables 5 through 8 illustrate the toxicity of TIC807 to westerntarnished plant bug (WTPB), Lygus hesperus and the tarnished plant bug(TPB), Lygus lineolaris. The diet was less than optimal for the TPB,reducing the rate of growth of the nymphs in the UTC (untreated check)sample relative to the UTC for WTPB. However, significant mortality andstunting was demonstrated against TPB.

TABLE 5 TIC807 stunting scores for western tarnished plant bug (WTPB),Lygus hesperus concentration Mean Standard Treatment (mg/ml) N stuntingDeviation P > |t| UTC 0.00 12 0.00 0.00 TIC807 1.00 5 1.60 0.55 <0.0001

TABLE 6 TIC807 per cent mortality scores for western tarnished plant bug(WTPB), Lygus hesperus concentration Mean % Standard Treatment (mg/ml) Nmortality Deviation P > |t| UTC 0.00 12 0.00 0.00 TIC807 1.00 5 56.7915.89 <0.0001

TABLE 7 TIC807 stunting scores for the tarnished plant bug (TPB), Lyguslineolaris concentration Mean Standard Treatment (mg/ml) N stuntingDeviation P > |t| UTC 0.00 6 0.00 0.00 TIC807 1.00 5 1.20 0.20 <0.05

TABLE 8 TIC807 per cent mortality scores for the tarnished plant bug(TPB), Lygus lineolaris concentration Mean % Standard Treatment (mg/ml)N mortality Deviation P > |t| UTC 0.00 6 0.00 0.00 TIC807 1.00 5 45.337.65 <0.05

Example 6 Synthesis of a Gene Encoding a TIC807 Protein that is Designedfor Expression in Plants

A nucleotide sequence encoding a TIC807 protein is designed andsynthesized. This non-native coding region designed for plant expressionis provided here as SEQ ID NO:6. The coding sequence is characterized bya lower A+T content than the native TIC807 coding region that wasderived from Bacillus thuringiensis, eliminating regions of the nativeTIC807 gene that are A+T rich and replacing those with sequences thathave fewer A+T residues.

Example 7 Expression Cassettes for Expression of a TIC807 Protein inTransgenic Plant Cells or Transgenic Plants

A variety of plant expression cassettes were constructed with thenon-native TIC807 coding region (SEQ ID NO:6). Such expression cassettesare useful for transient expression in plant protoplasts or plantcallus.

A first TIC807 plant expression cassette comprises an enhanced CaMV35Spromoter that is operably linked to a coding region comprising anon-native TIC807 encoding sequence with an in-frame C-terminal fusionto a myc-protein epitope tag (SEQ ID NO:19). This coding region isoperably linked to a 3′ terminal nopaline synthase (NOS) polyadenylationsite. The sequence of this non-targeted and tagged5′-e35S-TIC807-myc-NOS-3′ expression cassette is provided as (SEQ IDNO:20) and was cloned in pMON59221. The entire 5′-e35S-TIC807-myc-NOS-3′expression cassette is contained on a NotI restriction fragment in thepMON59221 shuttle vector.

A second TIC807 plant expression cassette comprises an enhanced CaMV35Spromoter that is operably linked to a coding region comprising anN-terminal Arabidopsis shkG chloroplast peptide encoding sequence (i.e.,CTP2) fused in frame to a non-native TIC807 encoding sequence with anin-frame C-terminal fusion to a myc-protein epitope tag (SEQ ID NO:21).This coding region is operably linked to a 3′ terminal nopaline synthase(NOS) polyadenylation site. The sequence of this targeted and taggede35S-CTP2-TIC807-myc-NOS expression cassette is provided as (SEQ IDNO:22) and was cloned in pMON59223. The entire5′-e35S-CTP2-TIC807-myc-NOS-3′ expression cassette is contained on aNotIrestriction fragment in the pMON59223 shuttle vector.

A third TIC807 plant expression cassette comprises an enhanced CaMV35Spromoter that is operably linked to a coding region comprising anon-native TIC807 encoding sequence (SEQ ID NO:6). This coding region isoperably linked to a 3′ terminal nopaline synthase (NOS) polyadenylationsite. The sequence of this non-targeted TIC807 expression cassette isprovided as (SEQ ID NO:40) and was cloned in pMON59224. The entire5′-e35S-TIC807-NOS-3′ expression cassette is contained on a NotIrestriction fragment in the pMON59224 shuttle vector.

A fourth TIC807 plant expression cassette comprises an enhanced CaMV35Spromoter that is operably linked to a coding region comprising anN-terminal Arabidopsis shkG chloroplast peptide encoding sequence (i.e.,CTP2) fused in frame to a non-native TIC807 encoding sequence (SEQ IDNO:7). The peptide sequence of the CTP2-TIC807 fusion protein encoded bythis construct is provided as SEQ ID NO:8. This coding region isoperably linked to a 3′ terminal nopaline synthase (NOS) polyadenylationsite. The sequence of this targeted 5′-e35S-CTP2-TIC807-NOS-3′expression cassette is provided as (SEQ ID NO:23) and was cloned inpMON59222. The entire 5′-e35S-CTP2-TIC807-NOS-3′ expression cassette iscontained on a NotI restriction fragment in the pMON59222 shuttlevector.

A fifth plastid-targeted expression cassette comprises an enhancedCaMV35S promoter that is operably linked to a 5′ untranslated leadersequence derived from the Glycine max Hsp17.9 gene which is operablylinked to a coding region comprising an N-terminal Arabidopsis shkGchloroplast peptide encoding sequence (i.e., CTP2) fused in frame to anon-native TIC807 encoding sequence (SEQ ID NO:6). The peptide sequenceof the CTP2-TIC807 fusion protein encoded by this construct is providedas SEQ ID NO:8. This coding region is operably linked to a 3′ terminalnopaline synthase (NOS) polyadenylation site. The sequence of thistargeted 5′-e35S-Hsp17.9-CTP2-TIC807-NOS-3′ expression cassette isprovided as (SEQ ID NO:42).

A sixth expression cassette comprises an enhanced CaMV35S promoter thatis operably linked to a 5′ untranslated leader sequence derived from theGlycine max Hsp17.9 gene which is operably linked to a coding regioncomprising a non-native TIC807 encoding sequence (SEQ ID NO:6). Thepeptide sequence of the TIC807 protein encoded by this construct isprovided as SEQ ID NO:5. This coding region is operably linked to a 3′terminal nopaline synthase (NOS) polyadenylation site. The sequence ofthis 5′-e35S-Hsp17.9-TIC807-NOS-3′ expression cassette is provided as(SEQ ID NO:43).

Example 8 Construction of Agrobacterium-Mediated Transformation VectorsContaining TIC807 Expression Cassettes and Transfer to Agrobacterium

To construct Agrobacterium mediated transformation vectors, TIC807expression cassettes are cloned into suitable vectors between theAgrobacterium border sequences such that they would be transferred tothe genome of a host plant cell by Agrobacterium hosts containing theconstructed vectors along with a selectable marker gene. Morespecifically, the restriction fragment containing the entire5′-e35S-Hsp17.9-CTP2-TIC807-NOS-3′ expression cassette (SEQ ID NO:42) iscloned into an Agrobacterium plant transformation vector. Similarly, therestriction fragment containing the entire 5′-e35S-Hsp17.9-TIC807-NOS-3′expression cassette (SEQ ID NO:43) is cloned into an Agrobacterium planttransformation vector. The vectors containing the TIC807 expressioncassettes (i.e., non-targeted cassette of SEQ ID NO:43 and targetedcassette of SEQ ID NO:42) are introduced into Agrobacterium byelectroporation or by tri-parental mating.

Example 9 Transformation of Cotton with TIC807 AgrobacteriumTransformation Vectors

Cotton can be transformed with the TIC807 Agrobacterium transformationvectors pMON105863 and pMON105864 or their equivalents using a proceduresubstantially similar to the procedure described in U.S. Pat. No.5,159,135.

To initiate the transformation and regeneration process for cottonplants, it is necessary to first surface sterilize cotton seeds toprevent inadvertent contamination of the resulting culture. The seedsare then allowed to germinate on an appropriate germinating mediumcontaining a fungicide.

Four to six days after germination the hypocotyl portion of the immatureplant is removed and sectioned into small segments averagingapproximately 0.5 centimeters apiece. The hypocotyl explants are allowedto stabilize and remain viable in a liquid or agar plant tissue culturemedium.

Once the hypocotyl segments have stabilized, they can promptly beinoculated with a suspension culture of transformation competentnon-oncogenic Agrobacterium. Agrobacterium strains such as LBA4404 canbe used. The inoculation process is allowed to proceed for three to fivedays at room temperatures, i.e., 24.degree. C.

At the end of the inoculation time period, it is necessary first torinse off the excess Agrobacterium. Then the remaining treated tissuescan be transferred to a second agar medium, which also contains one ormore antibiotics toxic to Agrobacterium, but not to hypocotyl tissues,at a concentration sufficient to kill any Agrobacterium remaining in theculture. Suitable antibiotics for use in such a medium includecarbenicillin and cefotaxime. The tissues are then given a period offrom one to ten days to recover from the transformation process and arethen continued in culture.

The tissues are now cultivated on a tissue culture medium which, inaddition to its normal components, contains a selection agent, theselection agent being one toxic to non-transformed cotton cells but notto transformed cotton cells which have incorporated genetic resistanceto the selection agent and are expressing that resistance.) A suitabletissue culture medium is the MS medium to which is added thephytohormones 2,4 dichlorophenoxy-acetic acid (2-4,D),6-furfurylaminopurine and a gelling agent. Suitable selection agentsinclude both antibiotics and herbicides. Suitable antibiotic traitswhich may serve as dominant selectable markers include theaminoglycoside phosphotransferase-3′-II (APH-(3′)-II) gene, alsoreferred to as the neomycin phosphotransferase II gene (NPTII), whichcode for resistance to the antibiotic kanamycin, and the APH-(3′)-IVgene which codes for resistance to Hygromycin B. Kanamycin, G418 andHygromycin B are aminoglycosides that will stop the growth ofnon-transformed cotton cells, but these antibiotics are phosphorylatedby the appropriate enzyme if it is expressed in the transformed cells.Another suitable selection agent is the herbicide glyphosate which canbe used to select for transformed cotton cells containing glyphosateresistant EPSPS genes. When using pMON105863 and pMON105864, thetransformed plant cells are selected for resistance to kanamycin oranother antibiotic that is closely related to kanamycin and inactivatedby the neomycin phosphotransferase II gene (NPTII) encoded by thesevectors. Antibiotic or herbicide dosed media allows only transformedcells to continue to grow and thrive. Thus the transformed cells, orcalli, are allowed to grow on the selective medium. The survivingtransformed tissues are transferred to a secondary medium to inducesomatic embryogenesis. The surviving transformed tissue will thuscontinue to form into somatic embryos, which can then be regeneratedthrough the regeneration technique of the present invention or throughany other alternative plant regeneration protocols which use cottonsomatic embryos as their starting point.

The selection process should continue for an extended time, i.e., 3-4months, because of the slow growth of even transformed tissues on theantibiotic medium. Subcultures are made every 4-6 weeks to replenishnutrients and antibiotics. As the transformed cells are selected andamplified, individually derived cell lines are identifiable and can beremoved and separately amplified.

The regeneration technique in accordance with the present inventionbegins with the tissues resulting from the transformation process. Thesetissues are putatively transformed calli which can generate somaticembryos when cultivated on appropriate embryo induction media. Onetechnique for regenerating these somatic embryos to whole plants isdisclosed here, but it is to be understood that other techniques arealso possible, once transformed embryogenic tissues are produced.

The regeneration technique used by the applicants here thus begins withthe tissues resulting from the transformation process. The cotton tissuecalli, generated from the hypocotyl segments of the cotton plants, andputatively transformed, are placed onto somatic embryo induction mediadirectly. At this point, the antibiotic selection agent should beremoved from the culture medium, but otherwise the medium may remainconstant. These calli, cultured on the somatic embryo induction medium,will form small embryoidal structures, which have been termed somaticembryos. It may take as long as two to three months for the somaticembryos to emerge and mature. Approximately 5 to as many as 20 somaticembryos will emerge from a single callus in an agar formulation of asomatic embryo induction medium. Many of the somatic embryos thusproduced will be regenerable into whole plants in accordance with thetechnique described here.

When the developing somatic embryos are large enough, i.e., to a size of4 mm or more in length, and if they appeared to have good embryonicdevelopment, i.e., usually having a cotyledon and a radicle, they may betransferred to large test tubes and hosted on fine vermiculite. Thevermiculite is saturated with Stewart and Hsu (SH) medium (Planta137:113 (1977)) plus the phytohormones indole acetic acid,6-furfurylaminpurine and gibberellic acid. Small plantlets, having twoto three leaves, eventually develop.

Once plantlet growth is established, i.e., the 2-3 leaf stage, theplants can now move into plant pots with vermiculite soil. They may bewatered and fertilized as needed. They may also need to be hardened off,before greenhouse exposure. The plantlets may be repotted when they have4-6 leaves after which they will continue to grow until mature. Samplesfrom the plantlets can be assayed for expression of TIC807 to identifytransgenic plants with insect inhibitory activity.

Example 11 In-Planta Testing of TIC807 in Callus Tissue

This example illustrates a non-limiting example of in planta expressionof TIC807 for bioassay against Lygus and other insect pests that pierceand/or suck the fluids from the cells and tissues of plants.

Cotton cells are transformed with constructs containing the TIC807protein encoding genes of interest. In this case, non-native A+T richnucleic acid sequences encoding a TIC807 protein are expressed in cottoncells using the TIC807 expression cassettes in the TIC807 transformationvectors described in the preceding examples. These expression cassettesprovide for either targeting of TIC807 to the chloroplast (i.e., withthe 5′-e35S-Hsp17.9-CTP2-TIC807-NOS-3′ or 5′-e35S-CTP2-TIC807-NOS-3′expression cassette) or non-targeted (cytoplasmic) expression of TIC807(i.e., with the 5′-e35S-Hsp17.9-TIC807-NOS-3′ or the5′-e35S-TIC807-NOS-3′ expression cassettes). The transformation vectorsprovide a selectable marker, in this case for selection of kanamycinresistance in transformed plant tissue. Either the pMON105863 vector orother equivalent TIC807 plant expression vectors that contain TIC807plant expression cassettes and a selectable marker can be used. Callustissue is allowed to develop in tissue culture after transformation andselection in a Petri dish. The Lygus nymphs are then placed into a Petridish or microtiter plate well containing callus that is transformed witha TIC807 plant expression cassette. Lygus nymphs are also placed into aPetri dish or microtiter plate well containing control callus that isnot transformed with a TIC807 plant expression cassette. The secured lidof the Petri dish or microtiter plate well prevents the escape of theLygus nymphs. Any material that will prevent Lygus escape but allow gasexchange in the Petri dish, for example, Parafilm® can be used to securethe Petri dish lid or microtiter plate well. A percentage of Lygusnymphs will find the callus tissue and feed. Scores for mortality andstunting are then calculated taking into account the background deaththat will occur from those insects which fail to feed on the callustissue to obtain an adjusted score. The adjusted scores for the Lygusnymphs presented with the TIC807 transformed tissue are compared withthe adjusted scores for the Lygus nymphs presented with control tissue.Scores for mortality and/or stunting for the Lygus nymphs presented withthe TIC807 transformed tissue are significantly increased relative tothe scores for the Lygus nymphs presented with control tissue.

Example 12 In-Planta Testing of TIC807 in Leaf Tissue

Alfalfa, cotton, canola, soybean, or lettuce cells are transformed usingthe TIC807 expression cassettes in the TIC807 transformation vectorsdescribed in the preceding examples. These expression cassettes providefor either targeting of TIC807 to the chloroplast (i.e., with the5′-e35S-CTP2-TIC807-myc-NOS-3′ or 5′-e35S-CTP2-TIC807-NOS-3′ expressioncassettes) or non-targeted (cytoplasmic) expression of TIC807 (i.e.,with the 5′-e35S-TIC807-myc-NOS-3′ or 5′-e35S-TIC807-NOS-3′ expressioncassettes). The transformation vectors provide a selectable marker, inthis case for selection of kanamycin resistance in transformed planttissue. The transformed cells are selected for resistance to kanamycinand regenerated into transgenic plants. Insect pests such as Lygusnymphs are then allowed to feed when the plant has reached a sufficientlevel of maturity, such as when the leaves have grown to a sizepermitting the use of a physical barrier to prevent Lygus escape. Thebarrier to prevent escape of the Lygus nymphs can be any commerciallyavailable or home made device that permits contact of the Lygus nymphswith the leaf tissue and allows the insect to probe and feed from thevascular tissue of the leaf. Clip cages similar to those described byMowry (1993) (J. Agric. Entomol. 10:181-184) would be sufficient tocontain the Lygus nymphs for feeding. Lygus nymphs are thus presentedwith leaf tissue from either transgenic plants that express the TIC807protein or with control leaf tissue that does not express TIC807protein. The control leaf tissue is ideally provided by a transgenicplant that was selected and regenerated in parallel but does not containa TIC-encoding transgene. However, leaf tissue from other plants ofsimilar origin and age can also be used so long as the tissue does notcontain significant amounts of TIC807 protein. Mortality and stuntingscores are then determined with respect to the background death thatwill occur from those insects which fail to feed on the leaf tissue toobtain an adjusted score. The adjusted scores for the Lygus nymphspresented with the TIC807 transformed leaf tissue are compared with theadjusted scores for the Lygus nymphs presented with control leaf tissue.Scores for mortality and/or stunting for the Lygus nymphs presented withthe TIC807 transformed leaf tissue are significantly increased relativeto the scores for the Lygus nymphs presented with control leaf tissue.

Example 13 Insect Control Using TIC807 in Combination with Other InsectControl Agents

Having obtained transgenic cotton plants that express either cytoplasmicor targeted TIC807 protein, it is also desirable to obtain cotton plantsthat express other insect inhibitory proteins in combination withTIC807. This example illustrates several methods by which increasedresistance to a specific insect pest or broader resistance to severalclasses of insect pests can be achieved to provide greater insectprotection for a crop plant. All of the strategies described below canalso be used to enhance an insect resistance management program.

I) Combination of TIC807 with Insect Inhibitory dsRNAi Molecules inPlants

Double stranded RNA mediated gene suppression of biological functionwithin target insect pests can be combined with genes encoding a TIC807protein. Insect genes that perform key biological functions that can betargeted by dsRNAi are described in U.S. Patent Application PublicationNo. US 2006/0021087.

Such dsRNAi molecules can be directed to inhibiting Lygus, which is thesame target insect that is inhibited by TIC807. By simultaneouslyinhibiting Lygus with a dsRNAi molecule and TIC807, inhibition isachieved through two distinct modes of action. Inhibition by distinctmodes of action is expected to result in improved insect resistancemanagement. For control of Lygus, dsRNAi molecules are derived from anyone of SEQ ID NO:4 through SEQ ID NO:39 that corresponds to genesexpressed in Lygus. The use of SEQ ID NO:24 through SEQ ID NO:39 in thecontrol of insects is disclosed in U.S. Patent Application PublicationNo. 20060021087. The dsRNAi molecules directed against any one of SEQ IDNO:24 through SEQ ID NO:39 are expressed in transgenic cotton plantswith Agrobacterium-mediated transformation vectors designed forexpression of ds RNAi molecules. Expression of dsRNAi molecules isachieved by recovery of transgenic plants comprising a promoter activein those plants that is operably linked to fragments of the Lygussequences (i.e., SEQ ID NO:24 through SEQ ID NO:39) of at least 19-24nucleotides in length and the reverse complements of those sequences.

Such dsRNAi molecules also can be directed to other piercing suckinginsects such as aphids, hoppers, or whiteflies. For control of aphids,dsRNAi molecules derived from or homologous to sequences from Toxopteracitricida or Acyrthosiphon pisum from U.S. Patent ApplicationPublication No. 2006/0021087 are expressed in transgenic plants withAgrobacterium-mediated transformation vectors designed for expression ofds RNAi molecules. For control of hoppers, dsRNAi molecules derived fromor homologous to sequences from Homalodisca coagulate from PatentApplication Publication No. U.S. 2006/0021087 are expressed intransgenic plants with Agrobacterium-mediated transformation vectorsdesigned for expression of ds RNAi molecules. For control of whiteflies,suitable dsRNAi molecules can be expressed in transgenic plants withAgrobacterium-mediated transformation vectors designed for expression ofds RNAi molecules. Expression of dsRNAi molecules is achieved byrecovery of transgenic plants comprising a promoter active in thoseplants that is operably linked to fragments of the respective aphid,hopper, or whitefly sequences of at least 19-24 nucleotides in lengthand the reverse complements of those sequences.

Such dsRNAi molecules also can be directed to coleopteran pests. In thiscase, expression of the dsRNAi molecule in conjunction with TIC807provides for control of both a coleopteran pest and a dipteran pest in atransgenic plant. For control of the boll weevil, Anthonomus grandisBoheman, dsRNAi molecules derived from a V-ATPase A ortholog sequencedescribed in U.S. Patent Application Publication No. 2006/0021087 areexpressed in transgenic cotton plants with Agrobacterium-mediatedtransformation vectors designed for expression of ds RNAi molecules.Expression of dsRNAi molecules is achieved by recovery of transgeniccotton plants comprising a promoter active in those plants that isoperably linked to fragments of the boll weevil V-ATPase A of at least19-24 nucleotides in length and the reverse complements of thosesequences.

Such dsRNAi molecules also can be directed to Lepidopteran pests. Inthis case, expression of the dsRNAi molecule in conjunction with TIC807provides for control of both a Lepidopteran pest and a Dipteran pest ina transgenic plant. For control of the army worm, dsRNAi moleculesderived from a midgut-expressed Army worm sequence (i.e., Helicoverpaarmigera sequences from U.S. Patent Application No. 2006/0021087) areexpressed in transgenic cotton plants with Agrobacterium-mediatedtransformation vectors designed for expression of ds RNAi molecules.Expression of dsRNAi molecules is achieved by recovery of transgeniccotton plants comprising a promoter active in those plants that isoperably linked to fragments of the Armyworm sequence of at least 19-24nucleotides in length and the reverse complements of those sequences.

I) Combination of TIC807 with Insect Inhibitory Proteins Other ThanTIC807 in Plants

For the control of piercing sucking insects such as aphids, hoppers,Lygus, or whiteflies, several toxin molecules can be combined withTIC807 expression in planta for greater control. Such moleculesexpressed inplanta along with TIC807 may include: i) ET29, ET37 orTIC809 and TIC810, TIC812, TIC127 or TIC128 (PCT US 2006/033867; U.S.Pat. No. 6,093,695); ii) AXMI-027, AXMI-036, and/or AXMI-038 (WO06/107761); iii) AXMI-018, AXMI-020, and/or AXMI-021 (WO 06/083891); iv)AXMI-010 (WO 05/038032); v) AXMI-003 (WO 05/021585) vi) AXMI-008 (US2004/0250311); vii) AXMI-006 (US 2004/0216186) viii) AXMI-007 (US2004/0210965); ix) AXMI-009 (US 2004/0210964); x) AXMI-014 (US2004/0197917); xi) AXMI-004 (US 2004/0197916); xii) AXMI-028 and/orAXMI-029 (WO 06/119457) and xiii) AXMI-007, AXMI-008, AXMI-0080rf2,AXMI-009, AXMI-014 and AXMI-004 (WO 04/074462). The combination of thetoxin protein molecules TIC809 (presented as SEQ ID NO:10) and TIC810(presented as SEQ ID NO:12) has been previously shown to be inhibitoryto the Western Tarnished Plant Bug (WTPB), Lygus hesperus Knight inbioassay (PCT US 2006/033867). The fusion proteins of TIC809 and TIC810,TIC127 (presented as SEQ ID NO:14) and TIC128 (presented as SEQ IDNO:16) may also be active against Lygus. The polynucleotide encodingTIC127 is comprised of the nucleic acid molecule encoding TIC809 linkedto the nucleic acid molecule encoding TIC810 by a polylinker nucleotidesequence (presented as SEQ ID NO:17) encoding the amino acid linkerpresented as SEQ ID NO:18. The polynucleotide encoding TIC128 iscomprised of the nucleic acid molecule encoding TIC810 linked to thenucleic acid molecule encoding TIC809 by a polylinker nucleotidesequence (presented as SEQ ID NO:17) encoding the amino acid linkerpresented as SEQ ID NO:18. Expression of TIC807 in combination withTIC127 or TIC128 may provide enhanced control of Lygus. Dicot plantssuch as cotton could be transformed with plant expression constructscontaining dicot-optimized nucleotide sequences encoding TIC807(presented as SEQ ID NO:6) along with TIC809 (presented as SEQ ID NO:9)and TIC810 (presented as SEQ ID NO:11), or TIC127 (presented as SEQ IDNO:13), or TIC128 (presented as SEQ ID NO:15) to provide enhancedresistance to Lygus or broader specificity to species contained withinthe genus, Lygus. Optimal expression of the toxin molecules may requiretargeted expression such as to the chloroplast of the cells. This can beachieved by the addition of a transit peptide-encoding nucleic acidmolecule, well known in the art to direct the translated protein to thechloroplast of the cell, to the 5′ end of the nucleic acid moleculeencoding the toxins.

DNA sequences encoding the TIC807 expression cassettes can be combinedwith either one or both of DNA sequences that encode insect inhibitorydouble stranded RNA and/or insect inhibitory protein molecules otherthan TIC807. These DNA molecules can be combined either through directtransformation or breeding, or a combination thereof, to produce eliteor hybrid plant lines demonstrating enhanced resistance to Lygus.

The combination of an insecticidal protein or proteins with one or moredouble stranded RNA, all independently active against a Hemipteran pestsuch as Lygus, is preferred as it provides two different modes of action(resistance management), and results in unexpected synergistic effectsthat are not observed with either the insecticidal protein alone, thedouble stranded RNA alone, or combinations of two different insecticidalproteins, both active against a Hemipteran pests, or combinations of twodifferent dsRNA's, both active against a Hemipteran pests. In addition,the spectrum of resistance of the crop plant could be broadened tocontain resistance to additional classes of insect pests such asColeopteran, Lepidopteran or Dipteran pests in addition to a Hemipteranpest such as Lygus using both the expression of insect toxin proteinsand double stranded RNA molecules within the plant.

For control of Lepidopteran pests and Hemipteran pests in a transgenicplant, plants expressing both a TIC807 protein and one or more proteinsactive against Lepidopteran pests can be obtained. Methods of obtainingtransgenic plants that are express Lepidopteran-active proteins such asCry1A proteins (U.S. Pat. No. 5,880,275), Cry1B (U.S. patent applicationSer. No. 10/525,318), Cry1C (U.S. Pat. No. 6,033,874), Cry1F, Cry1A/Fchimeras (U.S. Pat. Nos. 7,070,982, 6,962,705, and 6,713,063), and aCry2Ab protein (U.S. Pat. No. 7,064,249) are well characterized. Plantsexpressing both a TIC807 protein and a Lepidopteran active protein canbe obtained by making crosses of individual transgenic plants thatexpress TIC807 or the Lepidopteran protein(s). Alternatively, planttransformation vectors that provide for expression of both TIC807 or oneor more Lepidopteran active protein(s) can be used to transform plants.

Example 14 Toxicity of Purified Crystal Spore Preps of TIC807 to Lygushesperus

Toxicity of TIC807 to Lygus hesperus was also tested using a purifiedcrystal spore prep. Parasporal crystals containing the TIC807 proteinwere partially purified by sucrose gradient centrifugation. A10×-concentrated spore-crystal preparation of the TIC807 protein wastreated with Benzonase™ (Novagen; 10 U/ml sample) to reduce sampleviscosity. The treated sample was allowed to sit overnight at 4 C.Sucrose gradients in Ultraclear™ or Polyclear™ tubes suitable for a SW28rotor were prepared: 10 mL steps of 79%, 70%, and 55% sucrose in 10 mMTris-HCl, 0.1 mM EDTA, 0.005% Triton X-100 (pH 7). Approximately 6-7 mLsample were loaded per gradient (tubes filled to ¼ inch from the top).The gradients were run at 18K overnight (16-18 hr) in a SW28 rotor at 4C. Crystals were pulled from either the 55-70% interface or the 70-79%interface. Crystals were diluted at least 5-fold in gradient buffer andpelleted by centrifugation (e.g. 8K for 20 min at 4° C.). The crystalpellets were resuspended in buffer and examined under a phase-contrastmicroscope to assess spore contamination. Purified crystals weresubsequently treated with 50 mM CAPS-NaOH (pH 11) and incubated at 37 Cuntil the suspension cleared. The solubilized protein was dialyzedagainst 25 mM sodium carbonate, 10 mM NaCl (pH 8.0), loaded onto aQ-Sepharose column equilibrated with the same buffer, and eluted using alinear 10 mM-500 mM NaCl gradient. The eluted protein was dialyzedagainst 25 mM sodium carbonate (pH 8.5). The protein was judged to behighly purified by SDS-PAGE analysis. This TIC807 protein preparationwas observed to cause significant mortality and stunting (massreduction) of Lygus hesperus nymphs in the feeding assay when comparedto the untreated check and are presented in Tables 9 and Table 10 below.

TABLE 9 TIC807 stunting scores for western tarnished plant bug (WTPB),Lygus hesperus concentration Mean Standard Treatment (mg/ml) N stuntingDeviation P > |t| UTC 0 8 0 0 TIC807 0.05 5 2.0 0 <0.0001

TABLE 10 TIC807 per cent mortality scores for western tarnished plantbug (WTPB), Lygus Hesperus Concentration Mean % Standard Treatment(mg/ml) N mortality Deviation P > |t| UTC 0 8 0 0 TIC807 0.05 5 39.016.7 0.0003

Similar results were obtained in feeding assays with Lygus lineolaris.

Example 15 Synthesis of Additional Genes Encoding a TIC807 Protein thatare Designed for Expression in Plants

Additional nucleotide sequences encoding a TIC807 protein were designedand synthesized. These non-native coding region designed for plantexpression are provided here as SEQ ID NO: 44 through SEQ ID NO: 53. Thecoding sequences presented as SEQ ID NO: 44 through SEQ ID NO: 53 arecharacterized by a lower A+T content than the native TIC807 codingregion that was derived from Bacillus thuringiensis, eliminating regionsof the native TIC807 gene that are A+T rich and replacing those withsequences that have fewer A+T residues. SEQ ID NO: 44 is the nativeTIC807 coding region with the initiating Methionine start codon changedfrom the bacterial start codon of “TTG” to the plant start codon, “ATG’.

Example 16 Additional Expression Cassettes for Expression of a TIC807Protein in Transgenic Plant Cells or Transgenic Plants

A seventh plastid-targeted expression cassette comprises an enhancedCaMV35S promoter that is operably linked to a 5′ untranslated leadersequence derived from the Glycine max Hsp17.9 gene which is operablylinked to a coding region comprising an N-terminal Arabidopsis shkGchloroplast peptide encoding sequence (i.e. CTP2) fused in frame to anon-native TIC807 encoding sequence (SEQ ID NO:6). The peptide sequenceof the CTP2-TIC807 fusion protein encoded by this construct is providedas SEQ ID NO:8. This coding region is operably linked to a 3′ terminalCaMV35S polyadenylation site (T-35S). The sequence of this targeted5′-e35S-Hsp17.9-CTP2-TIC807-T-35S-3′ expression cassette is provided asSEQ ID NO:54.

An eighth expression cassette comprises an enhanced CaMV35S promoterthat is operably linked to a 5′ untranslated leader sequence derivedfrom the Glycine max Hsp17.9 gene which is operably linked to a codingregion comprising a non-native TIC807 encoding sequence (SEQ ID NO:6).The peptide sequence of the TIC807 protein encoded by this construct isprovided as SEQ ID NO:5. This coding region is operably linked to a 3′terminal CaMV35S polyadenylation site. The sequence of this5′-e35S-Hsp17.9-TIC807-T-35S-3′ expression cassette is provided as SEQID NO:55.

A ninth plastid-targeted expression cassette comprises a SugarcaneBadnavirus (ScBV) promoter (U.S. Pat. No. 5,994,123) that is operablylinked to a 5′ untranslated leader sequence derived from the Glycine maxHsp17.9 gene which is operably linked to a coding region comprising anN-terminal Arabidopsis shkG chloroplast peptide encoding sequence (i.e.CTP2) fused in frame to a non-native TIC807 encoding sequence (SEQ IDNO:6). The peptide sequence of the CTP2-TIC807 fusion protein encoded bythis construct is provided as SEQ ID NO:8. This coding region isoperably linked to a 3′ terminal CaMV35S polyadenylation site. Thesequence of this targeted 5′-P-ScBV-Hsp 17.9-CTP2-TIC807-T-35S-3′expression cassette is provided as SEQ ID NO:56.

A tenth expression cassette comprises a Sugarcane Badnavirus promoter(ScBV) that is operably linked to a 5′ untranslated leader sequencederived from the Glycine max Hsp17.9 gene which is operably linked to acoding region comprising a non-native TIC807 encoding sequence (SEQ IDNO:6). The peptide sequence of the TIC807 protein encoded by thisconstruct is provided as SEQ ID NO:5. This coding region is operablylinked to a 3′ terminal CaMV35S polyadenylation site. The sequence ofthis 5′-P-ScBV-Hsp17.9-TIC807-T-35S-3′ expression cassette is providedas SEQ ID NO:57.

Example 17 Construction of Additional Agrobacterium-MediatedTransformation Vectors Containing TIC807 Expression Cassettes andTransfer to Agrobacterium

To construct Agrobacterium mediated transformation vectors, TIC807expression cassettes are cloned into suitable vectors between theAgrobacterium border sequences such that they would be transferred tothe genome of a host plant cell by Agrobacterium hosts containing theconstructed vectors along with a selectable marker gene. Morespecifically, the restriction fragment containing the entire5′-e35S-Hsp17.9-CTP2-TIC807-T-35S-3′ expression cassette (SEQ ID NO:54)is cloned into an Agrobacterium plant transformation vector to obtainpMON105863 (FIG. 2). Similarly, the restriction fragment containing theentire 5′-e35S-Hsp17.9-TIC807-T-35S-3′ expression cassette (SEQ IDNO:55) is cloned into an Agrobacterium plant transformation vector toobtain pMON105864 (FIG. 3). For expression using a different promoter,the restriction fragment containing the entire5′-P-ScBV-Hsp17.9-CTP2-TIC807-T-35S-3′ expression cassette (SEQ IDNO:56) is cloned into an Agrobacterium plant transformation vector toobtain pMON78892 (FIG. 4). Similarly, the restriction fragmentcontaining the entire 5′-P-ScBV-Hsp17.9-TIC807-T-35S-3′ expressioncassette (SEQ ID NO:57) is cloned into an Agrobacterium plant expressionvector to obtain pMON78893 (FIG. 5). The vectors containing the TIC807expression cassettes (i.e. non-targeted cassette of SEQ ID NO:55 and SEQID NO:57 and targeted cassette of SEQ ID NO:54 and SEQ ID NO:56) areintroduced into Agrobacterium by electroporation or by tri-parentalmating.

The binary plant transformation vectors contain a selectable marker(indicated as “Selectable Marker” in FIGS. 2 through 5) for selection oftransformed plant cells using the antibiotic Kanamycin. Antibioticselection using Spectinomycin is used for bacterial selection. This isindicated as “SPC/STR” in FIGS. 2 through 5 which is comprised of apromoter for Tn7 adenyltransferase, the coding region for a geneencoding 3″ (9) —O-aminoglycoside adenyltransferase (AAD) derived fromStaphylococcus aureus and the transcription terminator region from Tn7adenyltransferase conferring spectinomycin and streptomycin resistance.Two origins for bacterial replication are included in each plasmid, anorigin of replication for Agrobacterium tumefaciens replication(indicated as “Ec.oriV-RK2” in FIGS. 2 through 5) and an origin forreplication in Escherichia coli (indicated as “Ori-322” in FIGS. 2through 5). An Escherichia coli coding region encoding a repressorprimer used in conjunction with the E. coli replication origin isindicated as “Ec.rop” in FIGS. 2 through 5. The left and right bordersused for stable integration of the T-DNA into the plant genome areindicated as “LB” and “RB”, respectively in FIGS. 2 through 5. FIG. 5also shows an additional expression cassette used in which the TIC128toxin protein (PCT US 2006/033867) is expressed and is labeled, “TIC128Expression Cassette” in FIG. 5.

Example 18 Additional In-Planta Testing of TIC807 in Callus Tissue

This example illustrates additional non-limiting examples of in plantaexpression of TIC807 for bioassay against Lygus and other insect peststhat pierce and/or suck the fluids from the cells and tissues of plants.

Alfalfa, cotton, canola, soybean, or corn cells are transformed usingthe TIC807 expression cassettes in the TIC807 transformation vectorsdescribed in the preceding examples. These expression cassettes providefor either targeting of TIC807 to the chloroplast (i.e., with the5′-e35S-Hsp17.9-CTP2-TIC807-T-35S-3′ or5′-P-ScBV-Hsp17.9-CTP2-TIC807-T-35S-3′ expression cassette) ornon-targeted (cytoplasmic) expression of TIC807 (i.e., with the5′-e35S-Hsp17.9-TIC807-T-35S-3′ or the 5′-P-ScBV-Hsp17.9-TIC807-T-35S-3′expression cassettes). The transformation vectors provide a selectablemarker, in this case for selection for kanamycin resistance intransformed plant tissue. The transformed cells are selected forresistance to kanamycin and regenerated into transgenic plants. Insectpests such as Lygus nymphs are then allowed to feed when the plant hasreached a sufficient level of maturity, such as when the leaves havegrown to a size permitting the use of a physical barrier to preventLygus escape. The barrier to prevent escape of the Lygus nymphs can beany commercially available or home made device that permits contact ofthe Lygus nymphs with the leaf tissue and allows the insect to probe andfeed from the vascular tissue of the leaf. Clip cages similar to thosedescribed by Mowry (1993) (J. Agric. Entomol. 10: 181-184) would besufficient to contain the Lygus nymphs for feeding. Lygus nymphs arethus presented with leaf tissue from either transgenic plants thatexpress the TIC807 protein or with control leaf tissue that does notexpress TIC807 protein. The control leaf tissue is ideally provided by atransgenic plant that was selected and regenerated in parallel but doesnot contain a TIC-encoding transgene. However, leaf tissue from otherplants of similar origin and age can also be used so long as the tissuedoes not contain significant amounts of TIC807 protein. Mortality andstunting scores are then determined with respect to the background deaththat will occur from those insects which fail to feed on the leaf tissueto obtain an adjusted score. The adjusted scores for the Lygus nymphspresented with the TIC807 transformed leaf tissue are compared with theadjusted scores for the Lygus nymphs presented with control leaf tissue.Scores for mortality and/or stunting for the Lygus nymphs presented withthe TIC807 transformed leaf tissue are significantly increased relativeto the scores for the Lygus nymphs presented with control leaf tissue.

Example 19 In-Planta Testing of TIC807 in Lettuce Leaf Tissue

Lettuce cells are transformed using the TIC807 expression cassettes inthe TIC807 transformation vectors described in the preceding examples.These expression cassettes provide for either targeting of TIC807 to thechloroplast (i.e. with the 5′-e35S-Hsp17.9-CTP2-TIC807-T-35S-3′ or5′-P-ScBV-Hsp17.9-CTP2-TIC807-T-35S-3′ expression cassettes) ornon-targeted (cytoplasmic) expression of TIC807 (i.e. with the5′-e35S-Hsp17.9-TIC807-T-35S-3′ or the 5′-P-ScBV-Hsp17.9-TIC807-T-35S-3′expression cassettes). The transformation vectors provide a selectablemarker, in this case for selection of kanamycin resistance intransformed plant tissue. The transformed cells are selected forresistance to kanamycin and regenerated into transgenic plants.

Lettuce seeds are surface sterilized for 20 minutes in 1.2% sodiumhypochlorite solution followed by 3 washes in sterilized deionizedwater. The seeds are allowed to dry overnight in a Petri dish in alaminar flow hood. The seeds are then plated on 100 ml 0.5× Hoagland'ssalts (see Table 11 below) in phytatrays (Sigma, St. Louis, Mo., Catalogno: P1552) at a density of 60 seeds/tray. The seeds are grown under thelight at 22 to 23 degrees Celsius for 4 to 5 days with a 16 hourphotoperiod. Agrobacterium transformed with the plant transformationvector of interest are prepared by inoculating 10 mls of liquidMannitol-Glutamate/Luria medium with 100 microliters of bacterialsuspension. The medium is comprised of the following ingredients:

LB broth, Miller (Difco #044-017-3) 12.5 g Mannitol 5.0 g Monosodiumglutamate(glutamic acid) 1.16 g KH2PO4 0.25 g MgSO47H2O 0.10 g Biotin0.001 g Total volume 1000 ml pH to 7.00 and autoclave

The liquid culture is incubated on a gyratory shaker at 28 degreesCelsius for 24 hours. Five milliliters of the first overnight culturesare diluted with 15 milliliters of Tryptone Yeast Extract mediasupplemented with 40 mg/L Acetosyringone (5 grams of Tryptone, 3 gramsof Yeast Extract and 20 ml of 2 mg/mL Acetosyringone in total volume of1000 ml, pH 5.5 and autoclaved). This is then allowed to incubate on agyratory shaker at 28 degrees Celsius for 24 hours in the dark with 50mg/L kanamycin and 100 mg/L spectinomycin. One ml of overnight cultureis added to 19 milliliters of Tryptone Yeast Extract media and the 600nm wavelength optical density of the culture is adjusted to 0.08 to0.09.

Lettuce seedling cotyledons are cut at both the base and the tip andsoaked in the diluted Agrobacterium medium for 15 minutes. Thecotyledons are then plated on MSO-C medium without blotting and kept at22 to 23 degrees Celsius with a 16 hour photoperiod. Plates are sealedwith micropore tape. After 48 hours, cotyledons are transferred to MSO-Imedium in 100 mm×25 mm Petri dishes. Explants are subsequentlysubcultured at 7 and 14 days to MSO-I medium. As shoots develop they areexcised and transferred to MSO-SE medium. Shoots are transferred afterelongation to phytatrays containing 100 ml of MSO-SE medium. After 6 to8 weeks, developing shoots are transferred to Magenta boxes containing100 ml of MSO-R medium. In 7 to 14 days of incubation at 23 degreesCelsius, roots will begin to develop. The shoots are then transferred to3 inch pots containing soil and allowed to grow. The composition of theMSO mediums is shown in table 11.

TABLE 11 MSO medium components. 0.5 X Hoagland's Ingredients salt MSO-CMSO-I MSO-SE MSO-R MSO salts (minimal salts) 34.6 g 34.6 g 34.6 g 34.6 gHoagland's salt 0.8 g Naphthaleneacetic acid 0.1 ml 0.1 ml 0.05 ml (1mg/ml) Benzyl adenine (1 mg/ml) 0.1 ml 0.1 ml 0.01 ml Acetosyringone (2mg/ml) 20 ml Kanamycin (50 mg/ml) 2 ml 2 ml 2 ml Carbenicillin (250mg/ml) 2 ml 2 ml 2 ml Tissue culture grade agar 7.5 g 7.5 g 7.5 g 8 g 8g Total volume 1000 ml 1000 ml 1000 ml 1000 ml 1000 ml pH 5.7 5.7 5.75.7

The transgenic plants are self-fertilized and allowed to set seed or areused directly for testing. The leaves of the transformed lettuce plantsare used in a culture system to test against Lygus. Ten milliliters ofsterile plant growth media (Murashige & Skoog, Gamborg B5 vitamins, 3%sucrose and 1.5% agar) is added while in liquid state to sterile 50milliliter polypropylene conical tubes. The media is allowed to cool andset under sterile conditions. Once set, a sterile circular foam divider,approximately the diameter of the tube containing a small hole in themiddle is placed over the plant growth media. Young lettuce leaves areexcised with a sterile razor blade and rinsed in sterile deionizedwater, leaving a portion of the petiole attached to the leaf. Thepetiole of the excised lettuce leaf is inserted through the hole andallowed to make contact with the media. Ten newly hatched (<12 hourspost-hatch) Lygus nymphs are added to the tube and a foam stopper isused to close the tube to allow gas exchange. The tube is kept in anincubator set to 25 degrees Celsius with a 14:10 day:night photoperiod.Mortality and stunting scores are then determined with respect to thebackground death that will occur from those insects which fail to feedon the leaf tissue to obtain an adjusted score. The adjusted scores forthe Lygus nymphs presented with the TIC807 transformed leaf tissue arecompared with the adjusted scores for the Lygus nymphs presented withcontrol leaf tissue. Scores for mortality and/or stunting for the Lygusnymphs presented with the TIC807 transformed leaf tissue aresignificantly increased relative to the scores for the Lygus nymphspresented with control leaf tissue.

Example 20 Expression of TIC807 in Transgenic Lettuce and Demonstrationof in Planta Toxicity

This example demonstrates Lygus hesperus toxicity of the TIC807 proteinwhen expressed in Lettuce plants. Lettuce plants were transformed withthe plant transformation vector, pMON105863 which contains the TIC807expression cassette, 5′-e35S-Hsp17.9-CTP2-TIC807-T-35S-3′ (SEQ ID NO:54)by the method described in Example 18. A control line of lettuce wastransformed using a vector control in which only the selection cassettefor transformation was contained. F1 seed was produced from thetransformed plants. Plants were grown from F1 seed and sampled forexpression of the TIC807 protein. Protein expression levels weredetermined for the F1 progeny using standard ELISA methods and TIC807polyclonal antibody. Plants were selected for Lygus hesperus testingbased upon TIC807 protein expression levels. Leaves were sampled fromselected F1 progeny and tested for Lygus hesperus toxicity using theassay method described in Example 18. Data from selected transgeniclettuce lines are presented in Table 12. Line events A028 and A055performed significantly better than the control (i.e. the untransformedlettuce line SVR3606). Overall analysis of the lines expressing theTIC807 protein demonstrated significant mortality relative to thecontrol transformed plants.

TABLE 12 Mean ± standard error of percentage Lygus hesperus mortality onTIC807 transformed lettuce leaves 15 infested teneral nymphs (n = 16)six days after infestation. TIC807 Concentration Event (ppm) PercentageMortality A028 43.22 67.71 ± 5.55 P ≧ 0.05 A055 41.62 62.08 ± 5.83 P ≧0.05 A035 30.77 51.04 ± 2.73 A002 33.02 50.00 ± 5.51 A059 42.90 48.96 ±2.83 Control 0.00 28.33 ± 2.15

Example 21 TIC807 Ts toxic to Colorado Potato Beetle

This example illustrates the toxicity of the insect toxin moleculesTIC807 to the coleopteran, Colorado potato beetle (CPB), Leptinotarsadecemlineata. Bioassays with CPB were conducted using an artificial dietconsisting of 13.2 g/L agar (Serva 11393), 140.3 g/L Bio-Serve pre-mix(Bio-Serve, Frenchtown, N.J. Catalog #F9380B), 5 ml/L Potassiumhydroxide (18.3% w/w) and 1.25 ml/L formalin (37%). The diet wasdispensed in 200 microliter aliquots into wells of a 96-well plate anddried briefly prior to sample application. Twenty microliters of testsample were applied per well, with sterile water serving as theuntreated check (UTC). Plates were allowed to dry before adding insectlarvae. One neonate CPB larva was added per well with a fine paintbrush.Plates were sealed with mylar and ventilated using an insect pin. Fortylarvae were tested per treatment. The bioassay plates were incubated at27 degrees Celsius with 60% relative humidity in complete darkness for10 to 12 days. The plates were scored for larval mortality. Data wereanalyzed using JMP® 4 statistical software (SAS Institute, Cary, N.C.).TIC807 demonstrated mortality when fed to CPB. The mean percentmortality scores for TIC807 is presented in Table 13.

It is thus contemplated that TIC807 proteins of the invention can beused to control Coleopteran insects. Coleopteran insects controlled byTIC807 proteins of the invention include, but are not limited to,Colorado potato beetle, wire worm and boll weevil.

TABLE 13 TIC807 percent mortality scores for the Colorado potato beetle(CPB), Leptinotarsa decemlineata. concentration Mean % StandardTreatment (mg/ml) N mortality Deviation P > |t| UTC 0 3 13.10 1.03TIC807 0.5 3 68.45 23.03 0.0142

Example 22 Transformation of Cotton with TIC807 and Toxin Testing UsingWhole Cotton Plants

Cotton cells are transformed with constructs containing a TIC807 proteinencoding gene of interest. In this case, codon redesigned nucleic acidsequences encoding for TIC807 protein are expressed in cotton cellsusing the TIC807 expression cassettes in the TIC807 transformationvectors described in the preceding examples. These expression cassettesprovide for either targeting of TIC807 protein to the chloroplast (i.e.with the expression cassettes, 5′-e35S-Hsp17.9-CTP2-TIC807-T-35S-3′found in pMON105863 or 5′-P-ScBV-Hsp17.9-CTP2-TIC807-T-35S-3′ found inpMON78892) or non-targeted (cytoplasmic) expression of TIC807 protein(i.e. with the expression cassettes, 5′-e35S-Hsp17.9-TIC807-T-35S-3′found in pMON105864 or the 5′-P-ScBV-Hsp17.9-TIC807-T-35S-3′ found inpMON78893). The transformation vectors provide a selectable marker, inthis case for selection of kanamycin resistance in transformed planttissue. Either the pMON105863 vector (FIG. 2), the pMON105864 vector(FIG. 3) or the pMON78892 vector (FIG. 4) or the pMON78893 vector (FIG.5) or other equivalent TIC807 plant expression vectors that containTIC807 plant expression cassettes and a selectable marker can be used.

Bioassay on plants expressing the TIC807 toxin molecule can be performedusing an enclosed cotton branch assay. Cotton plants are grown to anearly bloom stage where several fruiting branches containing squares(i.e. immature cotton flowers) are available. Sleeves are prepared usingbreathable plastic sheets (Vilutis and Co. Inc., Frankfort, Ill.).Sleeves are made using a standard photography or arts and craft tackingiron to create a seam producing a bag with an approximate dimension of 5inches×5 inches×12 inches long. Terminal branches including at least onepre-bloom square and unfolded terminal leaf are inserted into the openend of the sleeve. Alternatively, bags can be set up to enclose bolls orother tissues if desired. The bag is closed around the branch using atwist tie. Leaves and squares below the desired enclosed tissue can beremoved to facilitate secure closer with the twist tie. The other end ofthe sleeve is left open to allow insect infestation. Lygus nymphs arecollected with an aspirator and 4 nymphs are put into a 2 dram shellvial. Initial mass of the nymphs is recorded for each vial containingthe nymphs. The tube is tapped gently to assure the nymphs are at thebottom of the tube and the cap of the tube is removed. The tube isplaced inside the sleeve exposing the nymphs to the cotton plant tissue.The open end of the sleeve is then closed using a twist tie. The insectsare allowed to remain in the sleeves and feed upon the enclosed cottonplant material for a specified number of days, After the specified time,the cotton branches are removed. The sleeves are carefully opened tocount the surviving nymphs. All nymphs are collected and weighed.Mortality and stunting scores are then determined with respect tonon-transformed control plants. The adjusted scores for the Lygus nymphspresented with the TIC807 transformed cotton tissue are compared withthe adjusted scores for the Lygus nymphs presented with control cottontissue that lacks the TIC807 protein. Scores for mortality and/orstunting for the Lygus nymphs presented with the TIC807 transformedcotton tissue are significantly increased relative to the scores for theLygus nymphs presented with control cotton tissue.

Example 23 Combining TIC807 Toxin with Nectariless Cotton

This example illustrates using the nectariless phenotype of cotton incombination with TIC807 protein expression to provide greater control ofan insect pest. Lack of nectaries has been identified as arising fromhomozygosity for recessive mutations at two duplicate loci in Gossypiumtomentosum (Meyer and Meyer, 1961, Crop Science, 1: 167-169). Crosseswith Gossypium hirsutum with Gossypium tomentosum demonstrated asignificant reduction in populations of cabbage loopers and cottonleafworms in caged experiments relative to ordinary varieties of cottonin which floral nectarines are present (Lukefahr and Rhyne, 1960, Econ.Entomol. 53: 242-244). This is presumably the direct result ofnectariless cotton lines being less palatable to the insect pest as wellas the lack of sustenance provided by the nectars. Multiple mechanismsof resistance may be particularly crucial in Gossypieae species becauseextrafloral nectaries can directly attract some herbivore species.Extrafloral nectaries in cultivated cotton can enhance the abundance ofor damage by several crop pests including lepidopterans and plant bugs(Trelease, 1879, Nectar; what it is, and some of its uses. In J. H.Comstock [ed.], Report upon cotton insects, 319-343. U.S. Department ofAgriculture Publication, U.S. Government Publication Office, Washington,D.C., USA. Lukefahr and Rhyne, 1960; Lukefahr et al., 1960, Journal ofEcon Entom 53: 516-518; Benschoter and Leal, 1974, Journal of Econ Entom67: 217-218; Schuster et al., 1976, Journal of Econ Entom 69: 400-402;Wilson and Wilson, 1976, Journal of Econ Entom 69: 623-624; Henneberryet al., 1977, Journal of Econ Entom 70: 797-799; Adjei-Maafo et al.,1983, Environ Entom 12: 353-358; Beach et al., 1985, Journal ofEntomological Science 20: 233-236; Smith, 1992, Advances in Agronomy 48:251-296; Summy and King, 1992, Crop Protection 11: 307-319), mainlybecause adults of these taxa consume extrafloral nectar.

Lines produced by crosses of G. hirsutum with G. tomentosum are selectedfor the presence of the nectariless phenotype and favorable agronomictraits. In other embodiments, lines obtained from the commercialgermplasm Stoneville 825 can be used as a source of germplasm comprisingthe nectariless phenotype. In one embodiment of the method, the selectednectariless lines are then transformed with the an expression cassetteencoding either a TIC807 protein, the TIC809/TIC810 proteins, the TIC128protein or combinations thereof, or any other toxin molecule directed toa pest of cotton in which the presence of nectaries act as an attractantto the insect pest. In another embodiment of the method, transgeneinserts comprising an expression cassette encoding either a TIC807protein, the TIC809/TIC810 proteins, the TIC128 protein or combinationsthereof, or any other toxin molecule directed to a pest of cotton inwhich the presence of nectaries act as an attractant to the insect pestare obtained in any suitable cotton germplasm and then introgressed intolines produced by crosses of G. hirsutum with G. tomentosum that havebeen selected for the presence of the nectariless phenotype andfavorable agronomic traits. Through breeding methods known to one ofordinary skill in the art, the transformant lines expressing thenectariless phenotypes are selected and maintained in subsequentgenerations to contain both the nectariless phenotype and the insecttoxin molecule.

Example 24 Comparison of the TIC807 and Cry51Aa Proteins

To identify conserved and non-conserved regions of the TIC807 (SEQ IDNO:5) and Cry51 Aa (SEQ ID NO: 59) proteins, an alignment of the twoproteins was created using ClustalW. The Cry51Aa protein was isolatedfrom Bt strain F14-1, has a reported identity of 22% to cry15Aa, and isreported to have activity against Bombyx mori, a lepidopteran insect(Huang et al., J. Invertebr. Pathol. 95 (3), 175-180, 2007). A sequencefor the cry51Aa1 gene (SEQ ID NO: 58) and the encoded Cry51Aa1 protein(SEQ ID NO: 59) was reported as the NCBI GenBank Accession numberDQ836184. The ClustalW comparison of TIC807 (SEQ ID NO:5) and Cry51Aa1demonstrate that the two proteins have an overall sequence identity ofabout 97.4% over a length of 301 amino acids (see FIG. 6 and Table 14).The GenBank accession number DQ836184 reports a deduced N-terminus forCry51Aa1 that corresponds to use of a putative ATG start codon thatwould result in a 309 amino acid primary translation product. Incontrast, the TIC807 protein has a distinct amino terminus based on anN-terminal peptide sequence of isolated TIC807 protein that wasdetermined by Edman degradation (see Example 2 and SEQ ID NO:1). A TTGinitiator methionine codon of SEQ ID NO:4 is apparently used to generatethe TIC807 protein of SEQ ID NO:5. Consequently, the reported Cry51Aa1protein comprises an additional three amino acids at its' N-terminusthat do not occur in TIC807 (SEQ ID NO:5). In Cry51Aa1, the amino acidresidue corresponding to phenylalanine 46 of TIC807 is substituted for aserine residue, the amino acid residue corresponding to tyrosine 54 ofTIC807 is substituted for a histidine residue, the amino acid residuecorresponding to serine 167 of TIC807 is substituted for an arginineresidue, the three (3) amino acid residues corresponding to histidine199 to Serine 201 of TIC807 are deleted, and the amino acid residuecorresponding to serine 217 of TIC807 is substituted for an asparagineresidue. This comparison of the TIC807 and Cry51Aa1 proteins is shown inFIG. 6.

TABLE 14 # Sequence 1 2 1 TIC807 — 97.4 (301) (SEQ ID NO: 5) 2 Cry51Aa197.4 (301) — (SEQ ID NO: 59)

Although the TIC807 protein shares significant sequence identity to theCry51Aa1 protein, the TIC807 protein surprisingly displayed nosignificant level of activity when fed to certain lepidopteran insects.Purified TIC807 protein was tested against European Corn Borer (ECB;Ostrinia nubilalis) and tobacco budworm (TBW: Heliothis virescens) andhad no activity against either of those lepidopteran insects. The samesample of TIC807 protein displayed activity against Colorado PotatoBeetle CPB but not Corn Rootworm (CRW; Diabroticus). Thus the absence ofactivity of the purified TIC807 against ECB and TBW is not due toinactivity of the purified TIC807 protein.

The present invention thus contemplates TIC807 proteins that are activeagainst hemipteran and coleopteran insects but are inactive againstlepidopteran insects. In certain embodiments, the TIC807 proteins of theinvention can comprise TIC807 proteins wherein the corresponding residue54 of the TIC807 protein is not a histidine, wherein the correspondingresidue 167 of the TIC807 protein is not an arginine, wherein the three(3) amino acid residues corresponding to histidine 199 to Serine 201 ofTIC807 are present, and/or wherein the corresponding residue 217 of theTIC807 is not an asparagine residue. In still other embodiments, theTIC807 protein of the invention can comprise a protein that has at least98% or at least 99% identity over a length of 301 amino acidscorresponding to amino acid residues 2 to 309 of SEQ ID NO:5.

Various patent and non-patent publications are cited herein, thedisclosures of each of which are, to the extent necessary, incorporatedherein by reference in their entireties. Documents cited herein as beingavailable from the World Wide Web at certain internet addresses are alsoincorporated herein by reference in their entireties. Certain biologicalsequences referenced herein by their “NCBI Accession Number” can beaccessed through the National Center of Biotechnology Information on theworld wide web at ncbi.nlm.nih.gov.

As various modifications could be made in the constructions and methodsherein described and illustrated without departing from the scope of theinvention, it is intended that all matter contained in the foregoingdescription or shown in the accompanying drawings shall be interpretedas illustrative rather than limiting. Thus, the breadth and scope of thepresent invention should not be limited by any of the above-describedexemplary embodiments, but should be defined only in accordance with thefollowing claims appended hereto and their equivalents.

What is claimed is:
 1. A recombinant DNA comprising a promoter thatfunctions in a plant cell, a polynucleotide which encodes an insectinhibitory protein, wherein said insect inhibitory protein comprises apolypeptide sequence that has at least 99% sequence identity to SEQ IDNO:5, and a 3′ non-translated region containing a polyadenylationsignal, wherein said promoter, said polynucleotide, and said 3′non-translated region are operably linked.
 2. The recombinant DNA ofclaim 1, wherein said polypeptide sequence has 100% identity to SEQ IDNO:5.
 3. The recombinant DNA of claim 1, wherein said insect inhibitoryprotein inhibits a Hemipteran insect, a Heteropteran insect, aLeptinotarsa sp. insect or a Homopteran insect.
 4. The recombinant DNAof claim 3, wherein said Hemipteran insect is Lygus.
 5. The recombinantDNA of claim 3, wherein said Homopteran insect is an aphid, a hopper, ora whitefly.
 6. The recombinant DNA of claim 4, wherein said insectinhibitory protein inhibits Lygus at a Lygus diet concentration of atleast 5 ppm of said protein in said diet.
 7. The recombinant DNA ofclaim 6, wherein said insect inhibitory protein inhibits Lygus at aLygus diet concentration of at least 50 ppm of said protein in saiddiet.
 8. The recombinant DNA of claim 7, wherein said insect inhibitoryprotein inhibits Lygus at a Lygus diet concentration of at least 250 ppmof said protein in said diet.
 9. The recombinant DNA of claim 8, whereinsaid insect inhibitory protein inhibits Lygus at a Lygus dietconcentration of at least 500 ppm of said protein in said diet.
 10. Therecombinant DNA of claim 1, wherein said polynucleotide comprises asequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO:46, SEQ ID NO:47,SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52,and SEQ ID NO:53.
 11. The recombinant DNA of claim 1, designed forexpression in plants.
 12. A transgenic plant or plant part derivedtherefrom comprising an insect inhibitory protein, wherein said insectinhibitory protein comprises a polypeptide sequence that has at least99% sequence identity to SEQ ID NO:5.
 13. A transformed host cellcomprising a polynucleotide which encodes an insect inhibitory protein,wherein said insect inhibitory protein comprises a polypeptide sequencethat has at least 99% sequence identity to SEQ ID NO:5.
 14. Thetransformed host cell of claim 13, wherein said host cell is a bacterialcell or a plant cell.
 15. A plant derived from the transformed host cellof claim 14, said plant comprising said polynucleotide.
 16. A seedproduced from the plant of claim 15, said seed comprising saidpolynucleotide.
 17. A progeny plant from the seed of claim 16, saidprogeny plant comprising said polynucleotide.